Combination therapy using dual inhibitor of c-MET and EGFR and IGF-1R inhibitor

ABSTRACT

A method of preventing and/or treating a cancer, the method including co-administering a dual inhibitor of c-Met and EGFR (hereinafter, ‘c-Met/EGFR dual inhibitor’) and an IGF-1R inhibitor to a subject in need thereof and a use of IGF-1R as a marker for resistance to a c-Met/EGFR dual inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No. 10-2014-0163791 filed on Nov. 21, 2014 in the Korean Intellectual Property Office, the entire disclosure of which is hereby incorporated by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted herewith and identified as follows: One 152,884 byte ASCII (Text) file named “722073_ST25-Revised.TXT,” created Jul. 6, 2017.

BACKGROUND OF THE INVENTION 1. Field

Provided is a method of preventing and/or treating a cancer, the method including co-administering a dual inhibitor of c-Met and EGFR (hereinafter, ‘c-Met/EGFR dual inhibitor’) and an IGF-1R inhibitor to a subject in need thereof and a method for using IGF-1R as a marker for resistance to a c-Met/EGFR dual inhibitor.

2. Description of the Related Art

It has been shown that resistance to a drug having a specific target is more common compared to resistance to a drug having multiple targets. In addition, it is known that the indications on which a targeting drug has a therapeutic effect when it is treated alone are limited. Therefore, co-administration of two or more targeting drugs can maximize the therapeutic effect in a subject by overcoming resistance caused by exclusive treatment with only one of the targeting drugs, and by can exhibiting a therapeutic effect for an indication in which one of the targeting drugs has no therapeutic effect. Such co-administration is expected to contribute to extending the scope of indications to be treated by target drugs and to overcoming resistance thereto.

Therefore, for more effective treatment of a disease, there remains a need to develop effective combination therapy targeting two or more targets.

BRIEF SUMMARY OF THE INVENTION

An embodiment provides a combination therapy targeting c-Met, EGFR, and IGF-1R.

Another embodiment provides a pharmaceutical composition for combination therapy for treating and/or preventing cancer, the composition including a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor, as active ingredients.

Another embodiment provides a kit for treating and/or preventing cancer, including a first pharmaceutical composition including a c-Met/EGFR dual inhibitor, a second pharmaceutical composition including an IGF-1R inhibitor, and a package container.

Another embodiment provides a method of treating and/or preventing cancer, the method including co-administering a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor to a subject in need thereof.

Another embodiment provides a pharmaceutical composition for overcoming resistance to a c-Met/EGFR dual inhibitor, the composition including an IGF-1R inhibitor.

Another embodiment provides a method of overcoming resistance to a c-Met/EGFR dual inhibitor, the method including administering an IGF-1R inhibitor together with a c-Met/EGFR dual inhibitor to a subject who has resistance to the c-Met/EGFR dual inhibitor.

Another embodiment provides a marker for predicting and/or examining (monitoring) an effect of a c-Met/EGFR dual inhibitor, including IGF-1R protein and/or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R. The marker may be used for determining the presence or absence of resistance to a c-Met/EGFR dual inhibitor.

Another embodiment provides a method for predicting and/or examining (monitoring) an effect of a c-Met/EGFR dual inhibitor, including measuring a level of IGF-1R protein and/or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R in a biological sample obtained from a subject. The method may be used for determining the presence or absence of resistance to a c-Met/EGFR dual inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides western blotting results showing levels of IGF-1R protein in lung cancer cell lines H820 and HCC827 and gastric cancer cell lines SNU5, SNU668, and MKN45, indicating a correlation between level of IGF-1R protein and efficacy of combined administration of an anti-c-Met/anti-EGFR bispecific antibody and linsitinib (an IGF-1R inhibitor; indicated as “IGF1Ri”).

FIG. 2 provides graphs showing cancer cell proliferation inhibiting effects of combined administration of an anti-c-Met/anti-EGFR bispecific antibody and linsitinib in gastric cancer cell lines SNU5 and SNU668.

FIG. 3A is a graph showing cancer cell proliferation inhibiting effects of combined administration of anti-c-Met/anti-EGFR bispecific antibody ME22S and linsitinib in lung cancer cell line HCC827, depending on the concentrations of ME22S.

FIG. 3B is a graph showing cancer cell proliferation inhibiting effects of combined administration of ME22S and linsitinib and administration of ME22S only or linsitinib only in lung cancer cell line HCC827 at the concentrations of 30 nM of ME22S and 10 μM of linsitinib.

FIG. 4A is a graph showing cancer cell proliferation inhibiting effects of combined administration of anti-c-Met/anti-EGFR bispecific antibody ME22S and linsitinib in gastric cancer cell line MKN45, depending on the concentrations of ME22S.

FIG. 4B is a graph showing cancer cell proliferation inhibiting effects of combined administration of ME22S and linsitinib and administration of ME22S only or linsitinib only in gastric cancer cell line MKN45 at the concentrations of 0.4 nM of ME22S and 10 μM of linsitinib.

FIG. 5 provides graphs showing cancer cell proliferation inhibiting effects of combined administration of ME22S and linsitinib in anti-c-Met antibody resistant cell line MKN45, which has resistance to ME22S as well as anti-c-Met antibody L3-1Y-IgG2 (//note: we will provide related data together with our final instruction).

FIG. 6 provides western blotting results showing the changes in activities of downstream proteins by combined administration of an anti-c-Met/anti-EGFR bispecific antibody and linsitinib.

FIG. 7 provides western blotting results showing the level of IGF-1R protein, when an anti-c-Met/anti-EGFR bispecific antibody and linsitinib are administered together or respectively.

DETAILED DESCRIPTION OF THE INVENTION

A c-Met/EGFR dual inhibitor, such as an anti-c-Met/anti-EGFR bispecific antibody, generally exhibits strong anticancer effects on cancers having a high expression level of c-Met, such as gastric cancer, lung cancer, and the like. However, when resistance to an anti-c-Met/anti-EGFR bispecific antibody is inherently present in a subject (e.g., a c-Met and/or EGFR resistant cancer cell) or acquired by repeated administration of the anti-c-Met/anti-EGFR bispecific antibody, the anti-c-Met/anti-EGFR bispecific antibody cannot exhibit its effect on the subject, even if the expression level of c-Met in the subject is high. In cells with a high level of c-Met expression which are resistant to an anti-c-Met/anti-EGFR bispecific antibody (e.g., does not exhibit its anticancer effect), the expression level of IGF-1R is considerably higher compared to cells on which an anti-c-Met/anti-EGFR bispecific antibody exhibits its effect (e.g., anticancer effect). Based thereon, IGF-1R can serve as a marker for determining the presence or absence of resistance to an anti-c-Met/anti-EGFR bispecific antibody and/or as a target for a drug for being co-administered together with an anti-c-Met/anti-EGFR bispecific antibody in a combination therapy.

By co-administering an anti-c-Met/anti-EGFR bispecific antibody and an IGFR1 inhibitor to simultaneously inhibit c-Met, EGFR, and IGF-1R, an anticancer effect can be achieved even in cases where such effect cannot be obtained when only one or two of the targets is inhibited. Thus, the applicable scope of an anti-c-Met/anti-EGFR bispecific antibody can be expanded and resistance to an anti-c-Met/anti-EGFR bispecific antibody can be overcome.

In addition, when a c-Met/EGFR dual inhibitor and an IGFR1 inhibitor are co-administered simultaneously inhibiting c-Met, EGFR, and IGF-1R, the total overall dose of inhibitor can be decreased compared to co-administering a c-Met inhibitor, an EGFR inhibitor, and an IGFR1 inhibitor, thereby achieving both convenience in administration and decreased side effects such as cytotoxicity to normal cells caused by administration of three inhibitors.

For example, cancer cells in which an anti-c-Met/anti-EGFR bispecific antibody, a c-Met inhibitor, and/or an EGFR inhibitor do not exhibit an effect (e.g., an anticancer effect), an anticancer effect can be achieved by co-administration with an IGF-1R inhibitor. Such co-administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor overcomes an innate (inherent) or acquired resistance to a c-Met/EGFR dual inhibitor, thereby expanding the scope of symptoms and/or diseases to which a c-Met/EGFR dual inhibitor can be applicable.

Herein, the term “co-administration” and “combined administration” may be used interchangeably (i.e., have the same meaning).

The “c-Met” or “c-Met proteins” refer to receptor tyrosine kinases that bind to hepatocyte growth factors (HGF). The c-Met proteins may be those derived from all kinds of species, particularly a mammal, for example, those derived from a primate such as human c-Met (e.g. NP_000236.2), monkey c-Met (e.g., Macaca mulatta, NP_001162100), and the like, or those derived from a rodent such as mouse c-Met (e.g., NP_032617.2), rat c-Met (e.g., NP_113705.1), and the like. These proteins may include, for example, polypeptides encoded by the nucleotide sequence identified as GenBank Accession Number NM_000245.2, or proteins encoded by the polypeptide sequence identified as GenBank Accession Number NM_000236.2, or extracellular domains thereof. The receptor tyrosine kinase c-Met is involved in several mechanisms including cancer incidence, cancer metastasis, cancer cell migration, cancer cell penetration, angiogenesis, etc.

The “EGFR (epidermal growth factor receptor)” is a member of the receptor tyrosine kinases (RTKs) of HER family. The binding of a ligand to the extracellular domain of EGFR induces receptor homo- or hetero dimerization with other ErbB receptors, which in turn results in intracellular self-phosphorylation of specific tyrosine residues. EGFR self-phosphorylation leads to downstream signal transduction networks including MAPK and PI3K/Akt activation which affects cell proliferation, angiogenesis and metastasis. Over-expression, gene amplification, mutation, or rearrangement of EGFR are frequently observed in several human malignant tumors and are related to poor prognosis of cancer treatment and bad clinical outcomes. The EGFR or HER2 may be derived (obtained) from mammals, for example, primates such as humans and monkeys, or rodents such as rats and mice. For instance, the EGFR may be polypeptides encoded by the nucleotide sequences (mRNA or cDNA) deposited under GenBank Accession Nos. JQ739160, JQ739161, JQ739162, JQ739163, JQ739164, JQ739165, JQ739166, JQ739167, NM_005228.3, NM_201284.1, NM_201282.1, or NM_201283.1.

IGF-1R (insulin-like growth factor 1 receptor) binds to IGF1 (insulin-like growth factor 1) or IGF2 (insulin-like growth factor 2), and binds to insulin at a low affinity. The binding of IGF1 and IGF-1R increases the activity of receptor tyrosine kinases and induces auto-phosphorylation of the receptors or phosphorylation of internal related materials, thereby performing signal transduction. IGF-1R promotes proliferation of normal cells, but it can cause abnormal growth of cells. The overexpression of IGF-1R may cause formation of neoplasm, and thus its importance in cancer therapy emerged. The IGF-1R protein may be obtained from any species; for example, it may be at least one selected from the group consisting of human IGF-1R (e.g., AAI43722.1 (coding gene (cDNA or mRNA): BC143721.1), NP_000866.1 (coding gene (cDNA or mRNA): NM_000875.4), NP_001278787.1 (coding gene (cDNA or mRNA): NM_001291858.1)), etc.), mouse IGF-1R (e.g., AAI38870.1 (coding gene (cDNA or mRNA): BC138869.1), AAI38869.1 (coding gene (cDNA or mRNA): BC138868.1), NP_034643.2 (coding gene (cDNA or mRNA): NM_010513.2), etc.), and the like, but not be limited thereto.

It has been shown that IGF-1R signaling induces resistance to cytotoxic therapy and has a high relation with resistance to EGFR-targeting therapy. In particular, high expression of IGF-1R from cancer cells surgically excised from a gastric cancer patient is related to poor results of cancer therapy. In 86 gastric cancer patients having the surgical operation, the survival time is relatively longer for patients with low expression levels of IGF-1R and EGFR in the excised gastric cancer tissue compared to patients with high expression levels of IGF-1R and EGFR. Therefore, IGF-1R is considered as one of the important targets contributing to cancer cell growth through cross-talk with other RTKs.

As used herein, the term “c-Met/EGFR dual inhibitor” may refer to any agent (e.g., a compound or composition) capable of inhibiting activity and/or expression of c-Met and EGFR simultaneously, or blocking c-Met- and EGFR-signaling simultaneously by inhibiting ligands thereof, thereby preventing, improving, alleviating, reducing, and/or treating a c-Met- and/or EGFR-associated disease (e.g., a disease associating with abnormal activation and/or overexpression of c-Met and/or EGFR, such as cancer) or its symptoms. In addition, the c-Met/EGFR dual inhibitor may refer to any substance capable of recognizing and/or binding to c-Met and EGFR simultaneously to degrade and/or inhibit the functions or expression thereof. For example, the c-Met/EGFR dual inhibitor may be an anti-c-Met/anti-EGFR bispecific antibody which recognizes and/or binds to c-Met and EGFR at the same time. The anti-c-Met/anti-EGFR bispecific antibody may bind to both c-Met and EGFR and induce degradation of c-Met and/or EGFR.

As used herein, the term “effect of the c-Met/EGFR dual inhibitor” may refer to an effect to prevent, improve, alleviate, reduce, and/or treat a c-Met- and/or EGFR-associated disease, such as a cancer. For example, the effect of the c-Met/EGFR dual inhibitor may refer to an effect of reducing (or inhibiting proliferation of) cancer cells or cancer tissues, killing cancer cells or cancer tissues, and/or inhibiting invasion and/or migration of cancer cells related to metastasis. In other words, the effect may refer to an anticancer effect such as inhibiting of cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.

The present disclosure suggests a combination therapy by combined administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor, having advantages to expand the scope of application of the c-Met/EGFR dual inhibitor to indications (diseases) on which the c-Met/EGFR dual inhibitor does not exhibit an effect when used alone, and overcome resistance to the c-Met/EGFR dual inhibitor or the IGF-1R inhibitor. Of course, due to such effect to overcome resistance to the c-Met/EGFR dual inhibitor and a synergistic effect of the c-Met/EGFR dual inhibitor and the IGF-1R inhibitor, the combined administration of the c-Met/EGFR dual inhibitor and the IGF-1R inhibitor leads to more effective therapeutic effect on a disease on which the c-Met/EGFR dual inhibitor or IGF-1R inhibitor exhibits the effect even when each of them is used alone, and/or a disease having no resistance either to a c-Met/EGFR dual inhibitor nor to IGF-1R inhibitor. Therefore, by such combined administration, the dose of each inhibitor can be reduced and/or the administration interval can be increase (i.e., the frequency of administration can be decreased), thereby reducing side effects in a subject to be administered with the inhibitors.

One embodiment provides a pharmaceutical composition for combination therapy for preventing and/or treating of a cancer, comprising or consisting essentially of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor as active ingredients.

The pharmaceutical composition for combination therapy may be a mixed formulation (e.g., a single composition comprising two active ingredients) of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor. The IGF-1R inhibitor and c-Met/EGFR dual inhibitor may be included in any amount that is pharmaceutically effective when used together. The composition thus formulated can be used for simultaneous administration of the two active ingredients.

Alternatively, each of the c-Met/EGFR dual inhibitor and the IGF-1R inhibitor can be formulated in a separate composition and the two active ingredients (or compositions) can be separately administered simultaneously or sequentially. For instance, a first pharmaceutical composition including a pharmaceutically effective amount of the IGF-1R inhibitor as an active ingredient and a second pharmaceutical composition including a pharmaceutically effective amount of the c-Met/EGFR dual inhibitor as an active ingredient can be administered simultaneously or sequentially. In the case of the sequential administration, any order of administration may be used.

Another embodiment provides a kit useful for preventing and/or treating a cancer, comprising or consisting essentially of a first pharmaceutical composition including an IGF-1R inhibitor as an active ingredient, a second pharmaceutical composition including a c-Met/EGFR dual inhibitor as an active ingredient, and a package container. The IGF-1R inhibitor and c-Met/EGFR dual inhibitor may be used in amounts that are pharmaceutically effective when combined. The pharmaceutically effective amount of each inhibitor may be determined by the skilled medical practitioner or medical researcher. The package container can be any container that holds or otherwise links the two compositions in individual containers together in a single unit (e.g., a box that holds both containers, or plastic wrap that binds both containers together), or the package container may be a single, divided container having at least two chambers that each hold one of the two compositions.

A method of combination therapy for preventing and/or treating a cancer also is provided. The method comprises or consists essentially of co-administering a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor to a subject in need of the prevention and/or treatment of cancer. The IGF-1R inhibitor and c-Met/EGFR dual inhibitor may be administered in amounts that are pharmaceutically effective when combined. The pharmaceutically effective amount of each inhibitor may be determined by the skilled medical practitioner or medical researcher. The method may further include, prior to the co-administration step, a step of identifying a subject in need of the prevention and/or treatment of cancer. The step of identifying may be conducted by any manner and/or method known to the relevant field for identifying whether or not a subject needs the prevention and/or treatment of cancer. For example, the step of identifying may include diagnosing a subject with cancer or identifying a subject who is diagnosed as a cancer subject, particularly a cancer associated with c-Met/EGFR expression.

Co-administration may be conducted by administering the two active ingredients (i.e., c-Met/EGFR dual inhibitor and IGF-1R inhibitor) simultaneously; for example, by administering a mixed formulation (e.g., single composition) of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor, as described herein. Alternatively, the c-Met/EGFR dual inhibitor and IGF-1R inhibitor can be administered separately. The co-administration may be conducted by a first step of administering a c-Met/EGFR dual inhibitor, and a second step of administering an IGF-1R inhibitor, wherein the first and the second administration steps may be conducted simultaneously or sequentially. In case of the sequential administration, the first step and the second step may be performed in any order. In the sequential administration the first step and the second step may be separated by any suitable time interval (e.g., about 1 second to about 24 hours, about 1 second to about 12 hours, or about 1 second to about 60 minutes (e.g., about 1 second to about 10 minutes); or 1-60 seconds, 1-60 minutes, 1-24 hours, or 1-7 days, 1-14 days, 7-14 days, 1-30 days, or so on). The c-Met/EGFR dual inhibitor and IGF-1R inhibitor may be administered in amounts that are pharmaceutically effective when combined, which the amount may be determined by the skilled medical practitioner or medical researcher.

The term “the pharmaceutically effective amount” as used in this specification refers to an amount of which each active ingredient can exert pharmaceutically significant effects (e.g., an amount sufficient to prevent or treat cancer in a subject).

The subject for administration may be any animal, for example a mammal including a primate such as a human or a monkey, or a rodent such as a mouse or a rat, or a cell or tissue separated therefrom, or a culture of the cell or tissue. In particular, the subject may be a mammal including a primate such as a human or a monkey, or a rodent such as a mouse or a rat, or a cell or tissue separated therefrom, or a culture of the cell or tissue, who has an innate resistance or acquired resistance to a c-Met/EGFR dual inhibitor by inducing repeated administration.

By combined administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor, a synergistic therapeutic effect can be achieved compared to administration of c-Met inhibitor or IGF-1R inhibitor alone. Furthermore, such synergistic therapeutic effect can be obtained for diseases (e.g., a cancer) in which each of the inhibitors does not exhibit its effect or which has resistance to each of the inhibitors.

The IGF-1R inhibitor may refer to any drug capable of targeting and/or inhibiting the IGF-1R protein and/or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R. In particular, the IGF-1R inhibitor may be at least one selected from the group consisting of a compound (small molecule compound or its pharmaceutically acceptable salt), a protein, a nucleic acid and the like, which target and/or inhibit the IGF-1R protein and/or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R (e.g., an antibody, an aptamer, siRNA, shRNA, microRNA). For example, the IGF-1R inhibitor may be at least one selected from the group consisting of linsitinib (OSI-906; 3-[8-Amino-1-(2-phenyl-7-quinolyl)imidazo[1,5-a]pyrazin-3-yl]-1-methyl-cyclobutanol), NVP-AEW541 (CAS#475489-16-8; 7-((1s,3s)-3-(azetidin-1-ylmethyl)cyclobutyl)-5-(3-(benzyloxy)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine), GSK1904529A (CAS#1089283-49-7; N-(2,6-difluorophenyl)-5-(3-(2-(5-ethyl-2-methoxy-4-(4-(4-(methylsulfonyl)piperazin-1-yl)piperidin-1-yl)phenylamino)pyrimidin-4-yl)H-imidazo[1,2-a]pyridin-2-yl)-2-methoxybenzamide), NVP-ADW742 (CAS#475488-23-4; 5-(3-(benzyloxy)phenyl)-7-((1r,3r)-3-(pyrrolidin-1-ylmethyl)cyclobutyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine), BMS-536924 (CAS#468740-43-4; 4-[[(2S)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]-3-[4-methyl-6-(4-morpholinyl)-1H-benzimidazol-2-yl]-2(1H)-pyridinone), figitumumab (CP-751,871), cixutumumab (IMC-A12), dalotuzumab (MK-0646), R1507 (fully human anti-IGF-1R antibody), XL-228 (CAS#898280-07-4), INSM-18 (nordihydroguaiaretic acid; NDGA; 4,4′-((2R,3S)-2,3-dimethylbutane-1,4-diyl)bis(benzene-1,2-diol)), BMS-754807 ((S)-1-(4-((5-cyclopropyl-1H-pyrazol-3-yl)amino)pyrrolo[2,1-f][1,2,4]triazin-2-yl)-N-(6-fluoropyridin-3-yl)-2-methylpyrrolidine-2-carboxamide), AG-1024 (Tyrphostin; CAS#65678-07-1; 2-(3-bromo-5-tert-butyl-4-hydroxybenzylidene)malononitrile), GSK1838705A (CAS#1116235-97-2; 2-(2-(1-(2-(dimethylamino)acetyl)-5-methoxyindolin-6-ylamino)-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-6-fluoro-N-methylbenzamide), PQ 401 (CAS#196868-63-0; N-(5-Chloro-2-methoxyphenyl)-N′-(2-methyl-4-quinolinyl)urea), and pharmaceutically acceptable salts thereof, but not limited thereto.

XL-228:

The c-Met/EGFR dual inhibitor may be any drug comprising an EGFR binding domain and a c-Met binding domain, thereby simultaneously recognizing and/or binding to EGFR and c-Met. For example, the c-Met/EGFR dual inhibitor nay be an anti-c-Met/anti-EGFR bispecific antibody comprising or consisting essentially of i) an anti-EGFR antibody or an antigen-binding fragment thereof, and ii) an anti-c-Met antibody or an antigen-binding fragment thereof. The antigen-binding fragment of anti-EGFR antibody or anti-c-Met antibody may be scFv, (scFv)2, scFv-Fc (scFv and Fc are fused to each other), Fab, Fab′, or F(ab′)2. Alternatively, the c-Met/EGFR dual inhibitor may comprise or consist essentially of i) an antibody mimetic protein specifically binding to EGFR (e.g., an anti-EGFR DARPin (designed ankyrin repeat protein)), and ii) an anti-c-Met antibody or an antigen-binding fragment thereof.

The anti-EGFR antibody or an antigen-binding fragment thereof may comprise or consist essentially of,

at least one heavy chain complementarity determining region (CDR) selected from the group consisting of CDR-H1 including the amino acid sequence of SEQ ID NO: 109, CDR-H2 including the amino acid sequence of SEQ ID NO: 110, and CDR-H3 including the amino acid sequence of SEQ ID NO: 111 or a heavy chain variable region including the at least one heavy chain complementarity determining region;

at least one light chain complementarity determining region selected from the group consisting of CDR-L1 including the amino acid sequence of SEQ ID NO: 112, CDR-L2 including the amino acid sequence of SEQ ID NO: 113, and CDR-L3 including the amino acid sequence of SEQ ID NO: 114 or a light chain variable region including the at least one light chain complementarity determining region;

a combination of the at least one heavy chain complementarity determining region and the at least one light chain complementarity determining region; or

a combination of the heavy chain variable region and the light chain variable region.

TABLE 1 Heavy chain CDR Light chain CDR CDR-H1 NYDMS CDR-L1 TGSSSNIGNNDVS (SEQ ID NO: 109) (SEQ ID NO: 112) CDR-H2 GISHSSGSKYYADSVKG CDR-L2 DDNKRPS (SEQ ID NO: 110) (SEQ ID NO: 113) CDR-H3 KDATPRPLKPFDY CDR-L3 GSWDASLNA (SEQ ID NO: 111) (SEQ ID NO: 114)

For example, the anti-EGFR antibody or an antigen-binding fragment thereof may comprise or consist essentially of a heavy chain variable region including the amino acid sequence of SEQ ID NO: 115 or SEQ ID NO: 117, a light chain variable region including the amino acid sequence of SEQ ID NO: 116 or SEQ ID NO: 118, or a combination thereof.

In a particular embodiment, the anti-EGFR antibody or an antigen-binding fragment thereof may be an anti-EGFR scFv including a heavy chain variable region including the amino acid sequence of SEQ ID NO: 115 or SEQ ID NO: 117, and a light chain variable region including the amino acid sequence of SEQ ID NO: 116 or SEQ ID NO: 118.

<SEQ ID NO: 115: a heavy chain variable region of an anti-EGFR antibody> EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLE WVSGISHSSGSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCAKDATPRPLKPFDYWGQGTLVTVSS

(wherein the sequences marked in bold type indicate CDRs, i.e., CDR-H1, CDR-H2, and CDR-H3, in sequence)

<SEQ ID NO: 116: a light chain variable region of an anti-EGFR antibody> QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNDVSWYQQLPGTAPK LLIYDDNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSW DASLNAYVFGGGTKLTVLG

(wherein the sequences marked in bold type indicate CDRs, i.e., CDR-L1, CDR-L2, and CDR-L3, in sequence)

<SEQ ID NO: 117: a heavy chain variable region of an anti-EGFR antibody> EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKCLE WVSGISHSSGSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCAKDATPRPLKPFDYWGQGTLVTVSS

(wherein the sequences marked in bold type indicate CDRs, i.e., CDR-H1, CDR-H2, and CDR-H3, in sequence)

<SEQ ID NO: 118: a light chain variable region of an anti-EGFR antibody> QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNDVSWYQQLPGTAPK LLIYDDNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSW DASLNAYVFGCGTKLTVLG

(wherein the sequences marked in bold type indicate CDRs, i.e., CDR-L1, CDR-L2, and CDR-L3, in sequence)

Alternatively, the anti-EGFR antibody or an antigen-binding fragment thereof may be at least one selected from the group consisting of cetuximab (Erbitux), panitumumab, an anti-EGFR antibody or an antigen-binding fragment thereof comprising a heavy chain variable region of SEQ ID NO: 121, a light chain variable region of SEQ ID NO: 123, or a combination thereof, and an anti-EGFR antibody or an antigen-binding fragment thereof comprising a heavy chain variable region of SEQ ID NO: 125, a light chain variable region of SEQ ID NO: 126, or a combination thereof.

Alternatively, the EGFR binding domain may be an anti-EGFR DARPin.

DARPin (designed ankyrin repeat protein) refers to an antibody mimetic protein having high specificity and high binding affinity to a target protein, which is prepared via genetic engineering. DARPin is originated from natural ankyrin protein, and has a structure comprising at least 2 ankyrin repeat motifs, for example, comprising at least 3, 4 or 5 ankyrin repeat motifs. The DARPin can have any suitable molecular weight depending on the number of repeat motifs. For example, the DARPins including 3, 4 or 5 ankyrin repeat motifs may have a molecular weight of about 10 kDa, about 14 kDa, or about 18 kDa, respectively. DARPin includes a core that provides structure and a target binding portion that resides outside of the core and binds to a target. The structural core includes a conserved amino acid sequence and the target binding portion includes an amino acid sequence that differs depending on the target. Due to similarity (such as target specificity) with an antibody, DARPin may be fused with an IgG antibody to prepare a novel type of bispecific antibody.

The DARPin may be one targeting EGFR, i.e., an anti-EGFR DARPin (or EGFR binding DARPin), which specifically binding to EGFR. The anti-EGFR DARPin may be any DARPin having DARPin's own unique structure and specifically binding to EGFR. For example, the anti-EGFR DARPin may be at least one selected from the group consisting of the following 4 anti-EGFR DARPins:

anti-EGFR DARPin-01 (SEQ ID NO: 127): dlgkklleaaragqddevrilmangadvnaddtwgwtplhlaayqg hleivevllkngadvnaydyigwtplhlaadghleivevllkngad vnasdyigdtplhlaahnghleivevllkhgadvnaqdkfgktafd isidngnedlaeilq anti-EGFR DARPin-67 (SEQ ID NO: 128): dlgkklleaaragqddevrilmangadvnatdndgntplhlsawig hleivevllkhgadvnaddllgmtplhlaadtghleivevllkyga dvnardtrgktplhlaardghleivevllkhdadvnaqdkfgktaf disidngnedlaeilq anti-EGFR DARPin-68 (SEQ ID NO: 129): dlgkklleaaragqddevrilmangadvnafdywgmtplhlaadng hleivevllkhgadvnasdnfgftplhlaafyghleivevllkhga dvnafdmwgntplhlaaqnghleivevllkngadvnaqdkfgktaf disidngnedlaeilq anti-EGFR DARPin-69 (SEQ ID NO: 130): dlgkklleaaragqddevrilmangadvnaddnagrtplhlaanfg hleivevllkngadvnakghhcntplhlaawaghleivevllkyga dvnadddegytplhlaadigdleivevllkygadvnawdmygrtpl hlaasaghleivevllkygadvnaqdkfgktafdisidngnedlae ilq

The anti-EGFR DARPin may comprise about 1 to about 10, about 1 to about 5, or about 1 to about 3 of anti-EGFR DARPin units, wherein each of the DARPin units may be independently selected from SEQ ID NOs: 127 to 130. When the DARPin comprise about 2 or more DARPin units, each of the DARPin units may be the same with or different from each other.

When the EGFR binding domain is an antigen-binding fragment of an anti-EGFR antibody comprising a heavy chain variable region and a light chain variable region of an anti-EGFR antibody (e.g., an anti-EGFR scFv), or DARPin comprising 2 or more DARPin units, the heavy chain variable region and light chain variable region, or each DARPin unit may be linked to each other directly (i.e., with no linker) or via a linker. The linker may be a peptide linker, and if two or more linkers are used, the linkers may be the same with or different from each other. The peptide linker may include about 1 to about 100 or about 2 to about 50 (e.g., about 5 to about 25, about 1 to about 10, or about 2 to about 5) amino acids, and the kinds of the amino acids included in the peptide linker may not have any limitation. For example, the peptide linker may include Gly, Asn and/or Ser residues, or may include neutral amino acids such as Thr and/or Ala. Amino acid sequences suitable for a peptide linker may be well known in the relevant art. The length of the peptide linker may be properly determined so that there is no negative effect on the function of the bispecific chimeric protein. For example, the peptide linker may include at least one amino acid selected from the group consisting of Gly, Asn, Ser, Thr, and Ala, wherein the total number of the amino acids in the linker may be 1 to 100, 2 to 50, or 5 to 25. In one embodiment, the peptide linker may be represented as (GGGGS)n (SEQ ID NO: 132), wherein “n” is an integer from 1 to 10 (e.g., an integer from 2 to 5).

The anti-c-Met antibody or an antigen-binding fragment thereof may be any type of antibody or antigen-binding fragment thereof, which are capable of specifically recognizing and/binding to c-Met. The antigen-binding fragment may be scFv, (scFv)2, scFv-Fc, Fab, Fab′ or F(ab′)2.

The anti-c-Met antibody may be any antibody or antigen-binding fragment that acts on c-Met to induce intracellular internalization and degradation of c-Met. The anti-c-Met antibody may be any one recognizing a specific region of c-Met, e.g., a specific region in the SEMA domain, as an epitope.

c-Met, a receptor for hepatocyte growth factor (HGF), may be divided into three portions: extracellular, transmembrane, and intracellular. The extracellular portion is composed of an α-subunit and a β-subunit which are linked to each other through a disulfide bond, and contains a SEMA domain responsible for binding HGF, a PSI domain (plexin-semaphorin-integrin homology domain) and an IPT domain (immunoglobulin-like fold shared by plexins and transcriptional factors domain). The SEMA domain of c-Met protein may have the amino acid sequence of SEQ ID NO: 79, and is an extracellular domain that functions to bind HGF. A specific region of the SEMA domain, that is, a region having the amino acid sequence of SEQ ID NO: 71, which corresponds to a range from amino acid residues 106 to 124 of the amino acid sequence of the SEMA domain (SEQ ID NO: 79) of c-Met protein, is a loop region between the second and the third propellers within the epitopes of the SEMA domain. The region acts as an epitope for the specific anti-c-Met antibody of the present invention.

The term “epitope” as used herein, refers to an antigenic determinant, a part of an antigen recognized by an antibody. In one embodiment, the epitope for the c-Met antibody or antigen binding fragment thereof may be a region including about 5 or more contiguous amino acid residues within the SEMA domain (SEQ ID NO: 79) of c-Met protein, for instance, about 5 to about 19 contiguous amino acid residues within the amino acid sequence of SEQ ID NO: 71. For example, the epitope may be a polypeptide having about 5 to about 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, wherein the polypeptide essentially includes the amino sequence of SEQ ID NO: 73 (EEPSQ) serving as an essential element for the epitope. For example, the epitope may be a polypeptide including, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

The epitope including the amino acid sequence of SEQ ID NO: 72 corresponds to the outermost part of the loop between the second and third propellers within the SEMA domain of a c-Met protein. The epitope including the amino acid sequence of SEQ ID NO: 73 is a site to which the antibody or antigen-binding fragment according to one embodiment most specifically binds.

Thus, the anti-c-Met antibody may specifically bind to an epitope which has about 5 to about 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, including SEQ ID NO: 73 as an essential element. For example, the anti-c-Met antibody may specifically bind to an epitope including the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

In one embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may comprise or consist essentially of:

at least one heavy chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-H1 including the amino acid sequence of SEQ ID NO: 4; (b) a CDR-H2 including the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 2, or an amino acid sequence having about 8-19 consecutive amino acids within SEQ ID NO: 2 including amino acid residues from the 3^(rd) to 10^(th) positions of SEQ ID NO: 2; and (c) a CDR-H3 including the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 85, or an amino acid sequence having about 6-13 consecutive amino acids within SEQ ID NO: 85 including amino acid residues from the 1^(st) to 6^(th) positions of SEQ ID NO: 85, or a heavy chain variable region including the at least one heavy chain complementarity determining region;

at least one light chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-L1 including the amino acid sequence of SEQ ID NO: 7, (b) a CDR-L2 including the amino acid sequence of SEQ ID NO: 8, and (c) a CDR-L3 including the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 86, or an amino acid sequence having 9-17 consecutive amino acids within SEQ ID NO: 89 including amino acid residues from the 1^(st) to 9^(th) positions of SEQ ID NO: 89, or a light chain variable region including the at least one light chain complementarity determining region;

a combination of the at least one heavy chain complementarity determining region and at least one light chain complementarity determining region; or

a combination of the heavy chain variable region and the light chain variable region.

Herein, the amino acid sequences of SEQ ID NOS: 4 to 9 are respectively represented by following Formulas I to VI, below: Xaa₁-Xaa₂-Tyr-Tyr-Met-Ser (SEQ ID NO: 4),  Formula I

wherein Xaa₁ is absent or Pro or Ser, and Xaa₂ is Glu or Asp, Arg-Asn-Xaa₃-Xaa₄-Asn-Gly-Xaa₅-Thr (SEQ ID NO: 5),  Formula II

wherein Xaa₃ is Asn or Lys, Xaa₄ is Ala or Val, and Xaa₅ is Asn or Thr, Asp-Asn-Trp-Leu-Xaa₆-Tyr (SEQ ID NO: 6),  Formula III

wherein Xaa₆ is Ser or Thr, Lys-Ser-Ser-Xaa₇-Ser-Leu-Leu-Ala-Xaa₈-Gly-Asn-Xaa₉-Xaa₁₀-Asn-Tyr-Leu-Ala (SEQ ID NO: 7)  Formula IV

wherein Xaa₇ is His, Arg, Gln, or Lys, Xaa₈ is Ser or Trp, Xaa₉ is His or Gln, and Xaa₁₀ is Lys or Asn, Trp-Xaa₁₁-Ser-Xaa₁₂-Arg-Val-Xaa₁₃ (SEQ ID NO: 8)  Formula V

wherein Xaa₁₁ is Ala or Gly, Xaa₁₂ is Thr or Lys, and Xaa₁₃ is Ser or Pro, and Xaa₁₄-Gln-Ser-Tyr-Ser-Xaa₁₅-Pro-Xaa₁₆-Thr (SEQ ID NO: 9)  Formula VI

wherein Xaa₁₄ is Gly, Ala, or Gln, Xaa₁₅ is Arg, His, Ser, Ala, Gly, or Lys, and Xaa₁₆ is Leu, Tyr, Phe, or Met.

In one embodiment, the CDR-H1 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24. The CDR-H2 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26. The CDR-H3 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85.

The CDR-L1 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33, and 106. The CDR-L2 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36. The CDR-L3 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 12, 13, 14, 15, 16, 37, 86, and 89.

In another embodiment, the antibody or antigen-binding fragment may comprise or consist essentially of:

a heavy chain variable region comprising a polypeptide (CDR-H1) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24, a polypeptide (CDR-H2) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26, and a polypeptide (CDR-H3) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85;

a light chain variable region comprising a polypeptide (CDR-L1) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33 and 106, a polypeptide (CDR-L2) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36, and a polypeptide (CDR-L3) including an amino acid sequence selected from the group consisting of SEQ ID NOS 12, 13, 14, 15, 16, 37, 86, and 89; or

a combination the heavy chain variable region and the light chain variable region.

In one embodiment, the anti-c-Met antibody or antigen-binding fragment may comprise or consist essentially of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 131, 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99, or 107, or a combination of the heavy chain variable region and the light chain variable region.

In one embodiment, the anti-c-Met antibody may be a monoclonal antibody. The monoclonal antibody may be produced by the hybridoma cell line deposited with Accession No. KCLRF-BP-00220, which binds specifically to the extracellular region of c-Met protein (refer to Korean Patent Publication No. 2011-0047698, the disclosure of which is incorporated in its entirety herein by reference). The anti-c-Met antibody may include all the antibodies defined in Korean Patent Publication No. 2011-0047698.

By way of further example, the anti-c-Met antibody or the antibody fragment may include:

a heavy chain including the amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 62 (wherein the amino acid sequence from amino acid residues from the 1^(st) to 17^(th) positions is a signal peptide), the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62, the amino acid sequence of SEQ ID NO: 64 (wherein the amino acid sequence from the 1^(st) to 17^(th) positions is a signal peptide), the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64, the amino acid sequence of SEQ ID NO: 66 (wherein the amino acid sequence from the 1^(st) to 17^(th) positions is a signal peptide), and the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66; and

a light chain including the amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 68 (wherein the amino acid sequence from the 1^(st) to 20^(th) positions is a signal peptide), the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68, the amino acid sequence of SEQ ID NO: 70 (wherein the amino acid sequence from the 1^(st) to 20^(th) positions is a signal peptide), the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70, and the amino acid sequence of SEQ ID NO: 108.

For example, the anti-c-Met antibody may be selected from the group consisting of:

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 108;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 108; and

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 108.

According to an embodiment, the anti-c-Met antibody may include a heavy chain including the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68, or a heavy chain including the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the sequence of SEQ ID NO: 108.

The polypeptide of SEQ ID NO: 70 is a light chain including human kappa (κ) constant region, and the polypeptide with the amino acid sequence of SEQ ID NO: 68 is a polypeptide obtained by replacing histidine at position 62 (corresponding to position 36 of SEQ ID NO: 68 according to kabat numbering) of the polypeptide with the amino acid sequence of SEQ ID NO: 70 with tyrosine. The production yield of the antibodies may be increased by the replacement. The polypeptide with the amino acid sequence of SEQ ID NO: 108 is a polypeptide obtained by replacing serine at position 32 of SEQ ID NO: 108 (corresponding to position 52 of SEQ ID NO: 68, which corresponds to position 27e according to kabat numbering in the amino acid sequence from amino acid residues 21 to 240 of SEQ ID NO: 68; positioned within CDR-L1) with tryptophan. By such replacement, antibodies and antibody fragments including such sequences exhibits increased activities, such as c-Met biding affinity, c-Met degradation activity, Akt phosphorylation inhibition, and the like.

Animal-derived antibodies produced by immunizing non-immune animals with a desired antigen generally invoke immunogenicity when injected to humans for the purpose of medical treatment, and thus chimeric antibodies have been developed to inhibit such immunogenicity. Chimeric antibodies are prepared by replacing constant regions of animal-derived antibodies that cause an anti-isotype response with constant regions of human antibodies by genetic engineering. Chimeric antibodies are considerably improved in an anti-isotype response compared to animal-derived antibodies, but animal-derived amino acids still have variable regions, so that chimeric antibodies have side effects with respect to a potential anti-idiotype response. Humanized antibodies have been developed to reduce such side effects. Humanized antibodies are produced by grafting complementarity determining regions (CDR) which serve an important role in antigen binding in variable regions of chimeric antibodies into a human antibody framework.

The anti c-Met antibodies, the anti-EGFR antibodies and antigen-binding fragments thereof may be mouse-derived antibodies, mouse-human chimeric antibodies, humanized antibodies, or human antibodies. The antibodies or antigen-binding fragments thereof may be isolated from a living body or non-naturally occurring. The antibodies or antigen-binding fragments thereof may be recombinant or synthetic. The antibodies may be monoclonal.

An intact antibody includes two full-length light chains and two full-length heavy chains, in which each light chain is linked to a heavy chain by disulfide bonds. The antibody has a heavy chain constant region and a light chain constant region. The heavy chain constant region is of a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ϵ) type, which may be further categorized as gamma 1 (γ1), gamma 2(γ2), gamma 3(γ3), gamma 4(γ4), alpha 1(α1), or alpha 2(α2). The light chain constant region is of either a kappa (κ) or lambda (λ) type.

As used herein, the term “heavy chain” refers to full-length heavy chain, and fragments thereof, including a variable region V_(H) that includes amino acid sequences sufficient to provide specificity to antigens, and three constant regions, C_(H1), C_(H2), and C_(H3), and a hinge. The term “light chain” refers to a full-length light chain and fragments thereof, including a variable region V_(L) that includes amino acid sequences sufficient to provide specificity to antigens, and a constant region C_(L).

The term “complementarity determining region (CDR)” refers to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of immunoglobulin. The heavy and light chains may respectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDR may provide contact residues that play an important role in the binding of antibodies to antigens or epitopes. The terms “specifically binding” and “specifically recognized” are well known to one of ordinary skill in the art, and indicate that an antibody and an antigen specifically interact with each other to lead to an immunological activity.

The term “hinge region” as used herein, refers to a region between CH1 and CH2 domains within the heavy chain of an antibody which functions to provide flexibility for the antigen-binding site.

When an animal antibody undergoes a chimerization process, the IgG1 hinge of animal origin is replaced with a human IgG1 hinge or IgG2 hinge while the disulfide bridges between two heavy chains are reduced from three to two in number. In addition, an animal-derived IgG1 hinge is shorter than a human IgG1 hinge. Accordingly, the rigidity of the hinge is changed. Thus, a modification of the hinge region may bring about an improvement in the antigen binding efficiency of the humanized antibody. The modification of the hinge region through amino acid deletion, addition, or substitution is well-known to those skilled in the art.

In one embodiment, the anti-c-Met antibody, the anti-EGFR antibody, or an antigen-binding fragment thereof may be modified by the deletion, insertion, addition, or substitution of at least one amino acid residue on the amino acid sequence of the hinge region so that it exhibit enhanced antigen-binding efficiency. For example, the antibody may include a hinge region including the amino acid sequence of SEQ ID NO: 100(U7-HC6), 101(U6-HC7), 102(U3-HC9), 103(U6-HC8), or 104(U8-HC5), or a hinge region including the amino acid sequence of SEQ ID NO: 105 (non-modified human hinge). In particular, the hinge region has the amino acid sequence of SEQ ID NO: 100 or 101.

In the anti-c-Met antibody, the portion of the light chain and the heavy chain excluding the CDRs, the light chain variable region, and the heavy chain variable region as defined above, for example the light chain constant region and the heavy chain constant region, may be those from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, and the like).

The term “antigen-binding fragment” used herein refers to fragments of an intact immunoglobulin including portions of a polypeptide including at least one CDR or antigen-binding regions having the ability to specifically bind to the antigen. In a particular embodiment, the antigen-binding fragment may be scFv, (scFv)₂, scFvFc, Fab, Fab′, or F(ab′)₂, but is not limited thereto.

Among the antigen-binding fragments, Fab that includes light chain and heavy chain variable regions, a light chain constant region, and a first heavy chain constant region C_(H1), has one antigen-binding site.

The Fab′ fragment is different from the Fab fragment, in that Fab′ includes a hinge region with at least one cysteine residue at the C-terminal of C_(H1).

The F(ab′)₂ antibody is formed through disulfide bridging of the cysteine residues in the hinge region of the Fab′ fragment. Fv is the smallest antibody fragment with only a heavy chain variable region and a light chain variable region. Recombination techniques of generating the Fv fragment are widely known in the art.

Two-chain Fv includes a heavy chain variable region and a light chain region which are linked by a non-covalent bond. Single-chain Fv generally includes a heavy chain variable region and a light chain variable region which are linked by a covalent bond via a peptide linker or linked at the C-terminals to have a dimer structure like the two-chain Fv. The peptide linker may be the same as described in the above, for example, those having the amino acid length of about 1 to about 100, about 2 to about 50, particularly about 5 to about 25, and any kinds of amino acids may be included without any restriction.

The antigen-binding fragments may be attainable using protease (for example, the Fab fragment may be obtained by restricted cleavage of a whole antibody with papain, and the F(ab′)₂ fragment may be obtained by cleavage with pepsin), or may be prepared by using a genetic recombination technique.

In one embodiment, the c-Met/EGFR dual inhibitor (such as an anti-c-Met/anti-EGFR bispecific antibody) may comprise 1) an anti-c-Met antibody or an antigen binding fragment thereof, and 2) an anti-EGFR antibody, an antigen binding fragment thereof, or an EGFR DARPin, which is linked to the C-terminus or N-terminus of the anti-c-Met antibody. For example, the C-terminus or N-terminus of the anti-EGFR antibody may be linked to the C-terminus of the anti-c-Met antibody (e.g., the C-terminus of a heavy chain of the anti-c-Met antibody) or the antigen binding fragment thereof. Alternatively, the C-terminus or N-terminus of the anti-EGFR antibody may be linked to the N-terminus of the anti-c-Met antibody or the antigen binding fragment thereof.

In the c-Met/EGFR dual inhibitor, in order to fully perform the anti-c-Met antibody's activity to mediate intracellular migration and degradation of c-Met proteins, it may be advantageous that the anti-c-Met antibody has its own intact antibody structure. In addition, in case of the EGFR binding domain such as an anti-EGFR antibody and the like, its specific recognition and binding to EGFR is important, and thus it will be fine that just an antigen-binding fragment recognizing EGFR is included in the dual inhibitor (e.g., the bispecific antibody). Therefore, the c-Met/EGFR dual inhibitor such as an anti-c-Met/anti-EGFR bispecific antibody may be those including a complete (full length) form of an anti-c-Met antibody (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4) type antibody; comprising two heavy chain and two light chains) and an antigen binding fragment of the anti-EGFR antibody (e.g., an anti-EGFR scFv) or an anti-EGFR DARPin linked to the C terminus of the anti-c-Met antibody.

In the c-Met/EGFR dual inhibitor, the c-Met binding domain such as anti-c-Met antibody or the antigen binding fragment thereof, and the EGFR binding domain such as the anti-EGFR antibody, the antigen binding fragment thereof, or the anti-EGFR DARPin, may be linked via a peptide linker, or they may be linked directly and without a linker. Furthermore, a heavy chain portion and a light chain portion within the antigen binding fragment, for example, a heavy chain variable region and a light chain variable region within the scFv fragment, may be linked via a peptide linker or without a linker. The peptide linker which links the anti-c-Met antibody or the antigen binding fragment thereof and the anti-EGFR antibody or the antigen binding fragment thereof, and the peptide linker which links the heavy chain portion and the light chain portion within the antigen binding fragment, may be identical or different. The peptide linker may be include about 1 to about 100 amino acid residues, particularly about 2 to about 50, and any kinds of amino acids may be included without any restrictions. The peptide linker may include for example, Gly, Asn and/or Ser residues, and also include neutral amino acids such as Thr and/or Ala. Amino acid sequences suitable for the peptide linker may be those known in the pertinent art. Meanwhile, a length of the peptide linker may be variously determined within such a limit that the functions of the fusion protein will not be affected. For instance, the peptide linker may be formed by including a total of about 1 to about 100, about 2 to about 50, or about 5 to about 25 of one or more selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one embodiment, the peptide linker may be represented as SEQ ID NO: 132.

In the pharmaceutical composition (mixed formulation, first and second pharmaceutical compositions, etc.) or methods described herein, the active ingredients (a c-Met/EGFR dual inhibitor or an IGF-1R inhibitor) may be provided (comprised or administered) along with at least one additive selected from the group consisting of a pharmaceutically acceptable carriers, diluents, and excipients.

The pharmaceutically acceptable carrier to be included in the composition or mixed formulation may be those commonly used for the formulation of antibodies, which may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. The pharmaceutical composition may further include one or more selected from the group consisting of a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and preservative.

The pharmaceutical composition or mixed formulation may be administered orally or parenterally. The parenteral administration may include intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration. Since oral administration leads to digestion of proteins or peptides, an active ingredient in the compositions for oral administration must be coated or formulated to prevent digestion in stomach. In addition, the compositions may be administered using an optional device that enables an active substance to be delivered to target cells.

The pharmaceutically effective amount of the pharmaceutical composition, the c-Met/EGFR dual inhibitor, or the an IGF-1R inhibitor for a single dose may be prescribed in a variety of ways, depending on factors such as formulation methods, administration manners, age of subjects, body weight, gender, pathologic conditions, diets, administration time, administration interval, administration route, excretion speed, and reaction sensitivity. For example, the pharmaceutically effective amount of the IGF-1R inhibitor for a single dose may be in ranges of about 0.001 to about 100 mg/kg, or about 0.02 to about 10 mg/kg, and the pharmaceutically effective amount of the c-Met inhibitor for a single dose may be in ranges of about 0.001 to 100 mg/kg, or about 0.02 to about 10 mg/kg, but not limited thereto.

The term “pharmaceutically effective amount” used herein refers to an amount of the active ingredient (i.e., the c-Met/EGFR dual inhibitor, or the an IGF-1R inhibitor) exhibiting effects in preventing or treating cancer, and may be properly determined in a variety of ways, depending on factors such as formulation methods, administration methods, age of patients, body weight, gender, pathologic conditions, diets, administration time, administration route, excretion speed, and reaction sensitivity.

The pharmaceutically effective amount for the single dose may be formulated into a single formulation in a unit dosage form or formulated in suitably divided dosage forms, or it may be manufactured to be contained in a multiple dosage container. For the kit, the pharmaceutically effective amount of the c-Met/EGFR dual inhibitor and the pharmaceutically effective amount of the IGF-1R inhibitor for the single dose (one-time administration) may be each contained in a package container as a base unit.

In case that the co-administration comprises the sequential performance of the first administration step of administering the pharmaceutically effective amount of the c-Met/EGFR dual inhibitor and the second administration step of administering the effective amount of the IGF-1R inhibitor, the administration may be simultaneous or sequential with an interval between the first administration step and the second administration step of about 1 second to about 24 hours, about 1 second to about 12 hours, or about 1 second to about 60 minutes (e.g., about 1 second to about 10 minutes), and their administration may occur in any order. Alternatively, the administration interval between the co-administrations that is defined as a period between the co-administration and the subsequent co-administration may be, but not limited to, 1-60 seconds, 1-60 minutes, 1-24 hours, or 1-7 days, 1-14 days, 7-14 days, 1-30 days, or so on.

The mixed formulation or the pharmaceutical compositions for co-administration may be a solution in oil or an aqueous medium, a suspension, a syrup, or an emulsifying solution form, or they may be formulated into a form of an extract, powders, granules, a tablet or a capsule, and they may further include a dispersing agent or a stabilizing agent for their formulation.

In embodiments where the c-Met/EGFR dual inhibitor is an anti-c-Met antibody or an antigen binding fragment thereof and an anti-EGFR antibody or an antigen binding fragment thereof, the pharmaceutically effective amount of the c-Met inhibitor as an active ingredient may be formulated into an immunoliposome. A liposome containing an antibody may be prepared using any methods well known in the pertinent field. The immunoliposome is a lipid composition including phosphatidylcholine, cholesterol, and polyethyleneglycol-derivated phosphatidylethanolamine, which may be prepared by a reverse phase evaporation method. For example, Fab′ fragments of an antibody may be conjugated to the liposome through a disulfide-exchange reaction. A chemical drug, such as doxorubicin, may further be included in the liposome.

The pharmaceutical composition and method for co-administration proposed in this disclosure can be used for preventing and/or treating a cancer. The cancer may be a cancer related to or characterized by overexpression and/or abnormal activation of c-Met and/or EGFR. The cancer may be a solid cancer or blood cancer. The cancer may be a cancer on which a c-Met/EGFR dual inhibitor such as an anti-c-Met/anti-EGFR bispecific antibody has no anticancer effect when treated alone (i.e., a cancer that is not sensitive (responsive) to a c-Met/EGFR dual inhibitor such as an anti-c-Met/anti-EGFR bispecific antibody in the absence of an IGF-1R inhibitor), or which has resistance to the c-Met/EGFR dual inhibitor, as well as a cancer on which the c-Met/EGFR dual inhibitor has anticancer effect when treated alone or which has no resistance to the c-Met/EGFR dual inhibitor. The cancer may also have a resistance to treatment with an IGF-1R inhibitor used alone, or the cancer may be sensitive to treatment with an IGF-1R inhibitor. For instance, the cancer may be, not limited to, at least one selected from the group consisting of squamous cell carcinoma, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, skin cancer, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancers, brain cancer, osteosarcoma and so on. In a particular embodiment, the cancer may a cancer having innate or acquired resistance to the c-Met/EGFR dual inhibitor. In addition, the cancer may be one selected from gastric cancer, lung cancer, and the like, which have high expression of IGF-1R, but not be limited thereto. The cancer may be a primary cancer or a metastatic cancer.

The prevention and/or treatment effects of the cancer may include effects of not only inhibiting the growth of the cancer cells but also inhibiting the ability of the cancer to migrate, invade healthy cells, and metastasize.

Another embodiment provides partners for combination therapy capable of improving the effect of each other and overcoming resistance to each. In particular, provided is a use of an IGF-1R inhibitor as a partner drug for co-administration with a c-Met/EGFR dual inhibitor, that is, the drug is suitable for use in a combination therapy using an c-Met/EGFR dual inhibitor, and allows the c-Met/EGFR dual inhibitor to exhibit the effect on a disease on which the c-Met/EGFR dual inhibitor has no effect when it administered alone or disease having resistance to the c-Met/EGFR dual inhibitor.

Therefore, an embodiment provides a pharmaceutical composition for improving the effect of a c-Met/EGFR dual inhibitor, which comprises an IGF-1R inhibitor. Another embodiment provides a method for improving the effect of a c-Met/EGFR dual inhibitor, comprising co-administering a c-Met/EGFR dual inhibitor together with an IGF-1R inhibitor.

The term “improving the effect of a c-Met/EGFR dual inhibitor” may comprise making the c-Met/EGFR dual inhibitor to have an effect on a disease (e.g., a cancer) on which the c-Met/EGFR dual inhibitor has no or only little effect when it is used alone and/or a disease (e.g., a cancer) having resistance to the c-Met/EGFR dual inhibitor. Therefore, the composition or method for improving the effect of a c-Met/EGFR dual inhibitor may a composition or method for overcoming (treating, reducing, and/or alleviating) resistance to the c-Met/EGFR dual inhibitor.

Another embodiment provides a biomarker for predicting and/or monitoring an effect of a c-Met/EGFR dual inhibitor, comprising at least one selected from the group consisting of IGF-1R protein and nucleic acids (full length DNA, cDNA, or mRNA) encoding IGF-1R. The nucleic acid encoding IGF-1R may be full length DNA, cDNA, or mRNA of nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R.

Another embodiment provides a composition or a kit for predicting and/or monitoring an effect of a c-Met/EGFR dual inhibitor, comprising a material interacting with at least one selected from the group consisting of IGF-1R protein and nucleic acids encoding IGF-1R.

Another embodiment provides a method for predicting and/or monitoring an effect of a c-Met/EGFR dual inhibitor, comprising measuring the level of at least one selected from the group consisting of IGF-1R protein and nucleic acids (full-length DNA, cDNA or mRNA) encoding IGF-1R in a biological sample from a subject (to be tested).

The term “predicting an effect of a c-Met/EGFR dual inhibitor” may refer to predicting (determining) whether or not the c-Met/EGFR dual inhibitor exhibit its effect on an individual subject by confirming a factor affecting the display of effect of the c-Met/EGFR dual inhibitor, such as the presence/absence of innate (inherent) resistance to the c-Met/EGFR dual inhibitor. When it is confirmed that a c-Met/EGFR dual inhibitor exhibits an effect on an individual subject, the subject may be determined as a subject suitable for application of the c-Met/EGFR dual inhibitor. Therefore, the biomarker, composition, kit, or method for predicting an effect of a c-Met/EGFR dual inhibitor may be a biomarker, composition, kit, or method for selecting a subject for application of the c-Met/EGFR dual inhibitor.

The term “monitoring an effect of a c-Met/EGFR dual inhibitor” may refer to monitoring whether the c-Met/EGFR dual inhibitor exhibits its effect well or not and/or resistance to the c-Met/EGFR dual inhibitor is induced (acquired) by administration of the c-Met/EGFR dual inhibitor or not. Therefore, the biomarker, composition, kit, or method for monitoring an effect of a c-Met/EGFR dual inhibitor may be a biomarker, composition, kit, or method for monitoring induction (or acquisition) of resistance to the c-Met/EGFR dual inhibitor in a subject administered with the c-Met/EGFR dual inhibitor.

In an embodiment, provided is a method for predicting an effect of a c-Met/EGFR dual inhibitor, selecting a subject for application of a c-Met/EGFR dual inhibitor, monitoring an effect of a c-Met/EGFR dual inhibitor, or monitoring induction (or acquisition) of resistance to a c-Met/EGFR dual inhibitor. In the resistance, since an anti-c-Met antibody is comprised in the c-Met/EGFR dual inhibitor, when a resistance to the anti-c-Met antibody exists, the c-Met/EGFR dual inhibitor may lose its effect. Therefore, the resistance may refer to a resistance to a c-Met/EGFR dual inhibitor and/or a resistance to an anti-c-Met antibody.

As described above, a high level of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample (e.g., cells, tissues, body fluid, etc.) obtained (separated) from a subject may indicate that the biological sample or the subject has resistance to an anti-c-Met antibody and/or a c-Met/EGFR dual inhibitor. Therefore, in the method for predicting an effect of a c-Met/EGFR dual inhibitor or selecting a subject for application of a c-Met/EGFR dual inhibitor, when at least one selected from IGF-1R or nucleic acids encoding IGF-1R is absent or present at a low level, it can be determined (predicted) that the c-Met/EGFR dual inhibitor will exhibit an effect well on the biological sample or the subject from which the biological sample is obtained, or that the biological sample or the subject from which the biological sample is suitable for application of the c-Met/EGFR dual inhibitor. Therefore, the method for predicting an effect of a c-Met/EGFR dual inhibitor or selecting a subject for application of a c-Met/EGFR dual inhibitor may further comprise, after the measuring step, determining (predicting) an effect of a c-Met/EGFR dual inhibitor in the subject, selecting a subject for application of a c-Met/EGFR dual inhibitor, or determining that the biological sample or the subject from which the biological sample is suitable for application of the c-Met/EGFR dual inhibitor, when at least one selected from IGF-1R and nucleic acids encoding IGF-1R is absent (not detected or not measured) or present at a low level.

As used herein, the term “at least one selected from IGF-1R or nucleic acids encoding IGF-1R is present at a low level” may refer to the level (amount) of at least one selected from IGF-1R or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R in a biological sample (test sample) from a subject (to be tested) is lower than that of a reference sample. The reference sample may refer to a biological sample (e.g., cells, tissues, body fluid, etc.) on which the c-Met/EGFR dual inhibitor does not exhibit its effect (for example, an anticancer effect such as inhibiting of cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.), or a biological sample (e.g., cells, tissues, body fluid, etc.) separated (obtained) from a subject on which the c-Met/EGFR dual inhibitor does not exhibit its effect (for example, an anticancer effect such as inhibiting of cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.). For example, the reference sample may be at least one selected from the group consisting of, but not be limited to, H1373 (ATCC, CRL-5866) lung cancer cell line, HCC1806 (ATCC, CRL-2335) breast cancer cell line, Caki-1 (ATCC, HTB-46) renal cancer cell line, SKBR3 (ATCC, HTB-30) breast cancer cell line, BT474 (ATCC, HTB-20) breast cancer cell line, HT-29 (ATCC, HTB-38) colon cancer cell line, LoVo (ATCC, CCL-229) colon cancer cell line, HCT116 (ATCC, CCL-247) colon cancer cell line, SW620 (ATCC, CCL-227) colon cancer cell line, Ls174T (ATCC, CL-188) colon cancer cell line, and cell lines wherein resistance to an anti-c-Met antibody and/or resistance to a c-Met/EGFR dual inhibitor is induced by repeated or continued administration of the anti-c-Met antibody and/or c-Met/EGFR dual inhibitor.

The method for predicting an effect of a c-Met/EGFR dual inhibitor or selecting a subject for application of a c-Met/EGFR dual inhibitor may further comprise, after the measuring step and prior to the determining step (if it is comprised), comparing the level (amount) of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample (test sample) from a subject of interest (to be tested), with that of a reference sample as described above. For this, the method for predicting an effect of a c-Met/EGFR dual inhibitor or selecting a subject for application of a c-Met/EGFR dual inhibitor may further comprise, prior to the comparing step, measuring the level (amount) of at least one selected from IGF-1R or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R in the reference sample.

The steps of measuring the level of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in the biological sample and the reference sample are performed simultaneously or sequentially in any order.

Alternatively, if the IGF-1R level, which is determined by immunohistochemistry (IHC) using a conventional anti-IGF-1R-antibody (e.g., SAB4300359 (SIGMA), or G11 (Ventana; Catalog Number: 790-4346), etc.), is 0 to +1, it can be determined that IGF-1R is present at a low level or absent.

The method for predicting an effect of a c-Met/EGFR dual inhibitor or selecting a subject for application of a c-Met/EGFR dual inhibitor may further comprise, after the measuring, comparing, or determining step, administering a c-Met/EGFR dual inhibitor to the subject, when at least one selected from IGF-1R and nucleic acids encoding IGF-1R is absent (not detected or not measured) or present at a low level compared to the reference sample, as described above.

In another embodiment, a method for monitoring an effect of a c-Met/EGFR dual inhibitor, comprising measuring the level (amount) of at least one selected from IGF-1R or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R in a biological sample from a subject who is administered with the c-Met/EGFR dual inhibitor (or after administration of a c-Met/EGFR dual inhibitor). As described above, the method for monitoring an effect of a c-Met/EGFR dual inhibitor includes monitoring whether resistance to an anti-c-Met antibody and/or a c-Met/EGFR dual inhibitor is induced (acquired) or not.

In the method of monitoring an effect of a c-Met/EGFR dual inhibitor, it can be determined that a c-Met/EGFR dual inhibitor exhibits its effect (for example, an anticancer effect such as inhibiting of cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.) in the subject or resistance to the c-Met/EGFR dual inhibitor is not induced in the subject, if the level (amount) of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample from the subject who is administered with the c-Met/EGFR dual inhibitor (or after administration of a c-Met/EGFR dual inhibitor) is equal to (maintained) or decreased compared to that of a biological sample from a subject who is not administered with the c-Met/EGFR dual inhibitor (or before administration of a c-Met/EGFR dual inhibitor). Alternatively, it can be also determined that a c-Met/EGFR dual inhibitor does not exhibit its effect (for example, an anticancer effect such as inhibiting of cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.) in the subject or resistance to the c-Met/EGFR dual inhibitor is induced in the subject, if the level (amount) of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample from the subject who is administered with the c-Met/EGFR dual inhibitor (or after administration of a c-Met/EGFR dual inhibitor) is increased compared to that of a biological sample from a subject who is not administered with the c-Met/EGFR dual inhibitor (or before administration of a c-Met/EGFR dual inhibitor).

Therefore, the measuring step of the method of monitoring an effect of a c-Met/EGFR dual inhibitor may comprise measuring the levels (amount) of at least one selected from IGF-1R or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R in a biological sample from a subject before and after the subject is administered with a c-Met/EGFR dual inhibitor.

In addition, the method of monitoring an effect of a c-Met/EGFR dual inhibitor may further comprise, after the measuring step,

comparing the levels of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample from a subject before and after the subject is administered with a c-Met/EGFR dual inhibitor, and/or

determining that 1) the c-Met/EGFR dual inhibitor exhibits its effect (for example, an anticancer effect such as inhibiting of cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.) in the subject, a resistance to the c-Met/EGFR dual inhibitor is not induced (not acquired) in the subject, or the treatment using the c-Met/EGFR dual inhibitor can be maintained to the subject, when the level of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample from the subject after administration of a c-Met/EGFR dual inhibitor is equal to or decreased compared to that of a biological sample from a subject before administration of a c-Met/EGFR dual inhibitor, and/or 2) the c-Met/EGFR dual inhibitor does not exhibit its effect (for example, an anticancer effect such as inhibiting of cancer cell proliferation, migration (metastasis), or invasion, or inducing cancer cell apoptosis, etc.) in the subject, a resistance to the c-Met/EGFR dual inhibitor is induced (acquired) in the subject, or the treatment using the c-Met/EGFR dual inhibitor is stopped to the subject, when the level of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample from the subject after administration of a c-Met/EGFR dual inhibitor is increased compared to that of a biological sample from a subject before administration of a c-Met/EGFR dual inhibitor. The c-Met/EGFR dual inhibitor may be an anti-c-Met/anti-EGFR bispecific antibody.

The method of monitoring an effect of a c-Met/EGFR dual inhibitor may further comprise, after the measuring, comparing, or determining step, maintaining the administration a c-Met/EGFR dual inhibitor to the subject, when at least one selected from IGF-1R or nucleic acids encoding IGF-1R after a c-Met/EGFR dual inhibitor administration is equal to or decreased compared to that of before the c-Met/EGFR dual inhibitor administration.

In addition, as described above, when an IGF-1R inhibitor is co-administered with a c-Met/EGFR dual inhibitor, the effect of the c-Met/EGFR dual inhibitor can be improved (for example, resistance to an anti-c-Met antibody and/or a c-Met/EGFR dual inhibitor can be overcome); and thus, even if at least one selected from the group consisting of IGF-1R and a nucleic acid encoding IGF-1R exists in a biological sample from a subject, a desired effect can be achieved by co-administration of an IGF-1R inhibitor and a c-Met/EGFR dual inhibitor.

Therefore, another embodiment provides a method for predicting an effect of a combined administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor, and/or selecting a patient for application of a combined administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor, the method comprising measuring a level of at least one selected from the group consisting of IGF-1R and a nucleic acid encoding IGF-1R in a biological sample from a subject. As described above, the detection (presence) of at least one selected from the group consisting of IGF-1R and a nucleic acid encoding IGF-1R in a biological sample from a subject may indicate that the biological sample or the subject has innate (inherent) or acquired resistance to an anti-c-Met antibody and/or a c-Met/EGFR dual inhibitor; however, co-administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor can overcome such resistance, thereby achieving an increased therapeutic effect despite such resistance.

Therefore, the method for predicting an effect of a combined administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor and/or selecting a subject for application of a combined administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor, may further comprise predicting that the combined administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor is effective on the biological sample or the subject from which the biological sample is obtained or determining that the biological sample or the subject from which the biological sample is obtained is suitable for application of the combined administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor, if at least one selected from the group consisting of IGF-1R and a nucleic acid encoding IGF-1R is detected (present) in a biological sample from a subject. In an embodiment, the biological sample or biological sample or the subject from which the biological sample is obtained may have a resistance to an anti-c-Met antibody and/or a c-Met/EGFR dual inhibitor, which is innate (inherently) or acquired by repeated administration of a c-Met/EGFR dual inhibitor, wherein such innate (inherently) or acquired resistance to a c-Met/EGFR dual inhibitor can be overcome by co-administration of the c-Met/EGFR dual inhibitor together with an IGF-1R inhibitor.

In the above described method for predicting or monitoring an effect of a c-Met/EGFR dual inhibitor or a combined administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor or selecting a subject for application of a c-Met/EGFR dual inhibitor or a combined administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor, the step of measuring the level of at least one selected from IGF-1R or nucleic acids encoding IGF-1R (e.g., the step of measuring the level of at least one selected from IGF-1R or nucleic acids encoding IGF-1R in a biological sample from a subject and a reference sample or in a biological sample from a subject before and after the subject is administered with a c-Met/EGFR dual inhibitor may comprise i) applying (adding) an interacting material that interacts with at least one selected from the group consisting IGF-1R or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R to the biological sample and/or a reference sample; and ii) quantitatively analyzing the resulting reaction mixture to determine a level (amount) of at least one selected from the group consisting of IGF-1R or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R.

In an embodiment, prior to the step i), a step of providing a biological sample (and/or a reference sample) may be further performed. The step of providing a biological sample may comprise obtaining (isolating) a biological sample from a subject or obtaining a biological sample which has been isolated from a subject.

In step i), as will be further elucidated below, the interacting material may be at least one selected from the group consisting of compounds (a small molecular chemical; e.g., general label such as a fluorescent, a dye, etc.), proteins (antibodies, aptamers, etc.), nucleic acids (DNA, RNA, etc.), and the like, binding to IGF-1R or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R. For example, the interacting material may be at least one selected from the group consisting of a compound, an antibody, an aptamer, all specifically binding to IGF-1R, and a nucleic acid (e.g., a primer, a probe, an aptamer, etc.) binding to a part or entirety of a nucleic acid (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R, and optionally, may be conjugated with a label, such as a fluorescent or a dye. The step i) may be configured to form a complex by applying (adding) the interacting material to the biological sample (and/or a reference sample).

In step ii), the reaction mixture may be a complex resulting from interaction (binding) between at least one selected from the group consisting of IGF-1R and a nucleic acid (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R and the interacting material, which can be obtained in step i). The quantitatively analyzing step may comprise quantifying the complex, the label conjugated to the complex, or the IGF-1R or the nucleic acid gene segregated from the complex after the isolation of the complex from the biological sample (and/or the reference sample). The quantitative analysis of IGF-1R may be performed by any general quantifying means of proteins, such as immunochromatography, immunohistochemistry, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting, microarray, surface plasmon resonance (SPR), flow cytometry assay (e.g., Cytometric Bead Array (CBA) assay), Intracellular staining, Luminex assay, and the like, but not limited thereto. The quantitative analysis of nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding IGF-1R may be performed by any general quantifying means of genes (DNA or RNA), such as polymerase chain reaction (PCR; e.g., qPCR, RT-PCR, etc.), FISH (fluorescent in situ hybridization), microarray (e.g., mRNA microarray), and the like, but not limited thereto, which may use the interacting material such as a primer, a probe, or an aptamer, all capable of hybridizing (hybridizable) with a part or entirety of the nucleic acids encoding IGF-1R. In an embodiment, In one embodiment, the primer is designed to detect a fragment of successive base pairs out of the nucleic acid (full-length DNA, cDNA or mRNA) encoding IGF-1R, for example, a fragment of about 5 to about 2000 bp or about 5 to about 1000 bp, e.g., about 10 to about 1500 bp, about 10 to about 1000 bp, about 10 to about 500 bp, about 20 to about 200 bp, or about 50 to about 200 bp, and may be a pair of primers having nucleotide sequences which are respectively hybridizable with (e.g., complementary to) 3′- and 5′-terminal regions of the fragment, the region ranging in size from about 5 to about 100 bp, e.g., about 5 to about 50 bp, about 5 to about 30 bp, or about 10 to about 25 bp (that is, each of the primer pair comprises about 5 to about 100 nt, e.g., about 5 to about 50 nt, about 5 to about 30 nt, or about 10 to about 25 nt). The probe or the aptamer hybridizable with a part or entirety of the nucleic acid encoding IGF-1R may have a nucleotide sequence with a size from about 5 to about 2000 nt, from about 5 to about 1500 nt, from about 5 to about 1000 nt, from about 5 to about 500 nt, from about 5 to about 100 nt, from about 5 to about 50 nt, from about 5 to about 30 nt, or from about 5 to about 25 nt, which is hybridizable with (or complementary to) a fragment of the nucleic acid encoding IGF-1R. As used herein, the term “hybridizable” means pertaining to complementarily binding to a specific region of the nucleic acid, with a sequence complementarity of 80% or higher, e.g., 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100% between the primer, probe or aptamer and the nucleic acid region.

Considering that characteristic of the therapy using a c-Met/EGFR dual inhibitor such as an anti-c-Met/anti-EGFR bispecific antibody, a high level expression of c-Met in a cancer cell can serve as an advantageous condition so that a c-Met/EGFR dual inhibitor can more effectively exhibit its effect (e.g., an anticancer effect). Therefore, c-Met and/or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding c-Met may be serve as a marker for predicting or monitoring an effect of a c-Met/EGFR dual inhibitor or selecting a subject for application of a c-Met/EGFR dual inhibitor, and it may be used alone or in combination with IGF-1R and/or nucleic acid encoding IGF-1R.

Therefore, the composition for predicting or monitoring an effect of a c-Met/EGFR dual inhibitor or selecting a subject for application of a c-Met/EGFR dual inhibitor may further comprise an interacting material that interacts with at least one selected from the group consisting of c-Met and nucleic acids encoding c-Met, in addition to an interacting material that interacts with IGF-1R and/or nucleic acid encoding IGF-1R as described above. The interacting material that interacts with c-Met and/or nucleic acid encoding c-Met may be at least one selected from the group consisting of compounds (a small molecular chemical), proteins (antibodies, aptamers, etc.), nucleic acids (DNA, RNA, etc.), and the like, binding to c-Met or nucleic acids (e.g., full-length DNA, cDNA or mRNA) encoding c-Met. For example, the interacting material may be at least one selected from the group consisting of a compound, an antibody, an aptamer, all specifically binding to c-Met, and a nucleic acid (e.g., a primer, a probe, an aptamer, etc.) binding to a part or entirety of a nucleic acid (e.g., full-length DNA, cDNA or mRNA) encoding c-Met, and optionally, may be conjugated with a label, such as a fluorescent or a dye.

The method for predicting or monitoring an effect of a c-Met/EGFR dual inhibitor or selecting a subject for application of a c-Met/EGFR dual inhibitor may further comprise measuring a level of c-Met protein or a gene thereof (e.g., full-length DNA, cDNA, mRNA) in the biological sample from a subject. The steps of measuring the level of c-Met and/or c-Met coding nucleic acid and measuring the level of at least one selected from the group consisting of IGF-1R and nucleic acid encoding IGF-1R may be performed simultaneously or sequentially in any order. Details of the measuring step are as described above. For example, a western blotting technique may be employed. In this regard, when a predetermined amount (e.g., about 10 μg) of proteins obtained from a biological sample (e.g., cancer cells or tissues) is loaded on SDS PAGE gel, transferred onto the membrane, and reacted with an anti-c-Met antibody, and then, exposed on ECL reaction for a certain time (e.g., about 30 sec), the detection of a band may indicate that a prerequisite for the c-Met inhibitor therapy is established. In another embodiment, when a biological sample is found to have a c-Met mRNA level of about 13.5 or higher, about 13.6 or higher, or about 13.78 or higher, as measured by Affymetrix array (Affymetrix GeneChip Human Genome U133 Plus 2.0 array; using the primer set “203510_at”), a prerequisite for a c-Met/EGFR dual inhibitor therapy may be established. Cancer cells characterized by a high expression level of c-Met include cells from lung cancer, breast cancer, brain cancer, stomach cancer, liver cancer, and kidney cancer. However, any cancer cell, although derived from different kinds, may be a target of the c-Met inhibitor therapy if it expresses a high level of c-Met according to personal characteristics of patients.

In an embodiment, the biological sample used in the method for predicting or monitoring an effect of a c-Met/EGFR dual inhibitor or selecting a subject for application of a c-Met/EGFR dual inhibitor may be a tissue, a cell or body fluid (blood, serum, urine, saliva, etc.) which shows a high expression level of c-Met, for example, a c-Met level of about 13.5 or higher, about 13.6 or higher, or about 13.78 or higher, as measured by Affymetrix array (Affymetrix GeneChip Human Genome U133 Plus 2.0 array; using the primer set “203510_at”).

As used herein, the term “subject (suitable) for the application of a c-Met/EGFR dual inhibitor” means a patient to which a c-Met/EGFR dual inhibitor therapy is applicable suitably, and the term “subject” may be selected from the group consisting of mammals, such as rodents, e.g., mice, rats, etc., and primates, e.g., humans, monkeys, etc., e.g., a cancer patient. The biological sample may be a patient itself (mammals, such as primates, e.g., humans, monkeys, etc., or rodents, e.g., mice, rats, etc.) or a cell, a tissue or body fluid (e.g., blood, serum, urine, saliva, etc.) isolated from the patient or artificially an artificial culture thereof. The biological sample may be a cell, blood, or a serum.

Another embodiment provides a method of treating and/or preventing a cancer comprising co-administering a c-Met/EGFR dual inhibitor to a subject in need thereof. In the method of preventing and/or treating cancer, the subject may be one from whom a biological sample shows a low level (amount) of at least one selected from the group consisting of IGF-1R and a nucleic acid encoding IGF-1R compared to a reference sample, therein the term “low level” and “reference sample” are as described above. For example, the subject may be 1) selected by the above method for selecting a subject for the application of a combined administration of a c-Met/EGFR dual inhibitor.

Another embodiment provides a method of treating and/or preventing a cancer comprising co-administering a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor to a subject in need thereof. In the method of preventing and/or treating cancer, the subject may be one from whom a biological sample shows the presence of at least one selected from the group consisting of IGF-1R and a nucleic acid encoding IGF-1R. For example, the subject may be selected by the above method for selecting a subject for the application of a combined administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor.

The method of treating and/or preventing a cancer may further comprise identifying (selecting) the subject for application of a c-Met/EGFR dual inhibitor or application of a combined administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor, wherein the detailed process can be performed referring to the method of selecting a subject described above. The c-Met/EGFR dual inhibitor may be an anti-c-Met/anti-EGFR bispecific antibody.

In the above methods of treating and/or preventing a cancer, the c-Met/EGFR dual inhibitor and/or the IGF-1R inhibitor may be administered at a pharmaceutically effective amount as described above.

For example, the method of treating and/or preventing a cancer may comprise:

identifying a subject for application of a c-Met/EGFR dual inhibitor; and

administering a c-Met/EGFR dual inhibitor to the subject.

Alternatively, the method of treating and/or preventing a cancer may comprise:

identifying a subject for application of combined administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor; and

co-administering a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor to the subject.

Alternatively, the method of treating and/or preventing a cancer may comprise:

selecting a subject for application of a c-Met/EGFR dual inhibitor by measuring the level of IGF-1R and/or nucleic acid encoding IGF-1R in a biological sample from a subject; and

administering a c-Met/EGFR dual inhibitor to the subject.

Alternatively, the method of treating and/or preventing a cancer may comprise:

selecting a subject for application of combined administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor by measuring the level of IGF-1R and/or nucleic acid encoding IGF-1R in a biological sample from a subject; and

administering a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor to the subject.

In the above methods of treating and/or preventing a cancer, the step of selecting a subject may be performed referring to the above descriptions.

EXAMPLES

Hereafter, the present invention will be described in detail by examples.

The following examples are intended merely to illustrate the invention and are not construed to restrict the invention.

Reference Example 1: Construction of Anti-c-Met Antibody

1.1. Production of “AbF46”, a Mouse Antibody to c-Met

1.1.1. Immunization of Mouse

To obtain immunized mice necessary for the development of a hybridoma cell line, each of five BALB/c mice (Japan SLC, Inc.), 4 to 6 weeks old, was intraperitoneally injected with a mixture of 100 μg of human c-Met/Fc fusion protein (R&D Systems) and one volume of complete Freund's adjuvant. Two weeks after the injection, a second intraperitoneal injection was conducted on the same mice with a mixture of 50 μg of human c-Met/Fc protein and one volume of incomplete Freund's adjuvant. One week after the second immunization, the immune response was finally boosted. Three days later, blood was taken from the tails of the mice and the sera were 1/1000 diluted in PBS and used to examine a titer of antibody to c-Met by ELISA. Mice found to have a sufficient antibody titer were selected for use in the cell fusion process.

1.1.2. Cell Fusion and Production of Hybridoma

Three days before cell fusion, BALB/c mice (Japan SLC, Inc.) were immunized with an intraperitoneal injection of a mixture of 50 μg of human c-Met/Fc fusion protein and one volume of PBS. The immunized mice were anesthetized before excising the spleen from the left half of the body. The spleen was meshed to separate splenocytes which were then suspended in a culture medium (DMEM, GIBCO, Invitrogen). The cell suspension was centrifuged to recover the cell layer. The splenocytes thus obtained (1×10⁸ cells) were mixed with myeloma cells (Sp2/0) (1×10⁸ cells), followed by spinning to give a cell pellet. The cell pellet was slowly suspended, treated with 45% polyethylene glycol (PEG) (1 mL) in DMEM for 1 min at 37° C., and supplemented with 1 mL of DMEM. To the cells was added 10 mL of DMEM over 10 min, after which incubation was conducted in a water bath at 37° C. for 5 min. Then the cell volume was adjusted to 50 mL before centrifugation. The cell pellet thus formed was resuspended at a density of 1˜2×10⁵ cells/mL in a selection medium (HAT medium) and 0.1 mL of the cell suspension was allocated to each well of 96-well plates which were then incubated at 37° C. in a CO₂ incubator to establish a hybridoma cell population.

1.1.3. Selection of Hybridoma Cells Producing Monoclonal Antibodies to c-Met Protein

From the hybridoma cell population established in Reference Example 1.1.2, hybridoma cells which showed a specific response to c-Met protein were screened by ELISA using human c-Met/Fc fusion protein and human Fc protein as antigens.

Human c-Met/Fc fusion protein was seeded in an amount of 50 μL (2 μg/mL)/well to microtiter plates and allowed to adhere to the surface of each well. The antibody that remained unbound was removed by washing. For use in selecting the antibodies that do not bind c-Met but recognize Fc, human Fc protein was attached to the plate surface in the same manner.

The hybridoma cell culture obtained in Reference Example 1.1.2 was added in an amount of 50 μL to each well of the plates and incubated for 1 hour. The cells remaining unreacted were washed out with a sufficient amount of Tris-buffered saline and Tween 20 (TBST). Goat anti-mouse IgG-horseradish peroxidase (HRP) was added to the plates and incubated for 1 hour at room temperature. The plates were washed with a sufficient amount of TBST, followed by reacting the peroxidase with a substrate (OPD). Absorbance at 450 nm was measured on an ELISA reader.

Hybridoma cell lines which secrete antibodies that specifically and strongly bind to human c-Met but not human Fc were selected repeatedly. From the hybridoma cell lines obtained by repeated selection, a single clone producing a monoclonal antibody was finally separated by limiting dilution. The single clone of the hybridoma cell line producing the monoclonal antibody was deposited with the Korean Cell Line Research Foundation, an international depository authority located at Yungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 6, 2009, with Accession No. KCLRF-BP-00220 according to the Budapest Treaty (refer to Korean Patent Laid-Open Publication No. 2011-0047698).

1.1.4. Production and Purification of Monoclonal Antibody

The hybridoma cell line obtained in Reference Example 1.1.3 was cultured in a serum-free medium, and the monoclonal antibody (AbF46) was produced and purified from the cell culture.

First, the hybridoma cells cultured in 50 mL of a medium (DMEM) supplemented with 10% (v/v) FBS were centrifuged and the cell pellet was washed twice or more with 20 mL of PBS to remove the FBS therefrom. Then, the cells were resuspended in 50 mL of DMEM and incubated for 3 days at 37° C. in a CO₂ incubator.

After the cells were removed by centrifugation, the supernatant was stored at 4° C. before use or immediately used for the separation and purification of the antibody. An AKTA system (GE Healthcare) equipped with an affinity column (Protein G agarose column; Pharmacia, USA) was used to purify the antibody from 50 to 300 mL of the supernatant, followed by concentration with an filter (Amicon). The antibody in PBS was stored before use in the following examples.

1.2. Construction of chAbF46, a Chimeric Antibody to c-Met

A mouse antibody is apt to elicit immunogenicity in humans. To solve this problem, chAbF46, a chimeric antibody, was constructed from the mouse antibody AbF46 produced in Experimental Example 1.1.4 by replacing the constant region, but not the variable region responsible for antibody specificity, with an amino sequence of the human IgG1 antibody.

In this regard, a gene was designed to include the nucleotide sequence of “EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI” (SEQ ID NO: 38) for a heavy chain and the nucleotide sequence of “EcoRI-signal sequence-VL-BsiWI-CL-TGA-XhoI” (SEQ ID NO: 39) for a light chain and synthesized. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and a DNA fragment having the light chain nucleotide sequence (SEQ ID NO: 39) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen), and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invtrogen) (A), and in another 15 ml tube, 100 ul (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v) FBS for 5 hours at 37° C. under a 5% CO₂ condition and then in FBS-free DMEM for 48 hours at 37° C. under a 5% CO₂ condition.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a chimeric antibody AbF46 (hereinafter referred to as “chAbF46”).

1.3. Construction of Humanized Antibody huAbF46 from Chimeric Antibody chAbF46

1.3.1. Heavy Chain Humanization

To design two domains H1-heavy and H3-heavy, human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 purified in Reference Example 1.2 were analyzed. An Ig BLAST (www.ncbi.nlm.nih.gov/igblast/) result revealed that VH3-71 has an identity/identity/homology of 83% at the amino acid level. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VH3-71. Back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 30 (S→T), 48 (V→L), 73 (D→N), and 78 (T→L). Then, H1 was further mutated at positions 83 (R→K) and 84 (A→T) to finally establish H1-heavy (SEQ ID NO: 40) and H3-heavy (SEQ ID NO: 41).

For use in designing H4-heavy, human antibody frameworks were analyzed by a BLAST search. The result revealed that the VH3 subtype, known to be most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the VH3 subtype to construct H4-heavy (SEQ ID NO: 42).

1.3.2. Light Chain Humanization

To design two domains H1-light (SEQ ID NO: 43) and H2-light (SEQ ID NO: 44), human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 were analyzed. An Ig BLAST search result revealed that VK4-1 has a identity/homology of 75% at the amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VK4-1. Back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I). Only one back mutation was conducted at position 49 (Y→I) on H2-light.

To design H3-light (SEQ ID NO: 45), human germline genes which share the highest identity/homology with the VL gene of the mouse antibody AbF46 were analyzed by a search for BLAST. As a result, VK2-40 was selected. VL and VK2-40 of the mouse antibody AbF46 were found to have a identity/homology of 61% at an amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody were defined according to Kabat numbering and introduced into the framework of VK4-1. Back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H3-light.

For use in designing H4-light (SEQ ID NO: 46), human antibody frameworks were analyzed. A Blast search revealed that the Vk1 subtype, known to be the most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the Vk1 subtype. Back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H4-light.

Thereafter, DNA fragments having the heavy chain nucleotide sequences (H1-heavy: SEQ ID NO: 47, H3-heavy: SEQ ID NO: 48, H4-heavy: SEQ ID NO: 49) and DNA fragments having the light chain nucleotide sequences (H1-light: SEQ ID NO: 50, H2-light: SEQ ID NO: 51, H3-light: SEQ ID NO: 52, H4-light: SEQ ID NO: 53) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing a humanized antibody.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invtrogen) (A), and in another 15 ml tube, 100 ul (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a humanized antibody AbF46 (hereinafter referred to as “huAbF46”). The humanized antibody huAbF46 used in the following examples included a combination of H4-heavy (SEQ ID NO: 42) and H4-light (SEQ ID NO: 46).

1.4. Construction of scFv Library of huAbF46 Antibody

For use in constructing an scFv of the huAbF46 antibody from the heavy and light chain variable regions of the huAbF46 antibody, a gene was designed to have the structure of “VH-linker-VL” for each of the heavy and the light chain variable region, with the linker including the amino acid sequence “GLGGLGGGGSGGGGSGGSSGVGS” (SEQ ID NO: 54). A polynucleotide sequence (SEQ ID NO: 55) encoding the designed scFv of huAbF46 was synthesized in Bioneer and an expression vector for the polynucleotide had the nucleotide sequence of SEQ ID NO: 56.

After expression, the product was found to exhibit specificity to c-Met.

1.5. Construction of Library Genes for Affinity Maturation

1.5.1. Selection of Target CDRs and Synthesis of Primers

The affinity maturation of huAbF46 was achieved. First, six complementary determining regions (CDRs) were defined according to Kabat numbering. The CDRs are given in Table 2, below.

TABLE 2 CDR Amino Acid Sequence CDR-H1 DYYMS (SEQ ID NO: 1) CDR-H2 FIRNKANGYTTEYSASVKG(SEQ ID NO: 2) CDR-H3 DNWFAY (SEQ ID NO: 3) CDR-L1 KSSQSLLASGNQNNYLA (SEQ ID NO: 10) CDR-L2 WASTRVS (SEQ ID NO: 11) CDR-L3 QQSYSAPLT (SEQ ID NO: 12)

For use in the introduction of random sequences into the CDRs of the antibody, primers were designed as follows. Conventionally, N codons were utilized to introduce bases at the same ratio (25% A, 25% G, 25% C, 25% T) into desired sites of mutation. In this experiment, the introduction of random bases into the CDRs of huAbF46 was conducted in such a manner that, of the three nucleotides per codon in the wild-type polynucleotide encoding each CDR, the first and second nucleotides conserved over 85% of the entire sequence while the other three nucleotides were introduced at the same percentage (each 5%) and that the same possibility was imparted to the third nucleotide (33% G, 33% C, 33% T).

1.5.2. Construction of a Library of huAbF46 Antibodies and Affinity for c-Met

The construction of antibody gene libraries through the introduction of random sequences was carried out using the primers synthesized in the same manner as in Reference Example 1.5.1. Two PCR products were obtained using a polynucleotide covering the scFV of huAbF46 as a template, and were subjected to overlap extension PCR to give scFv library genes for huAbF46 antibodies in which only desired CDRs were mutated. Libraries targeting each of the six CDRs prepared from the scFV library genes were constructed.

The affinity for c-Met of each library was compared to that of the wildtype. Most libraries were lower in affinity for c-Met, compared to the wild-type. The affinity for c-Met was retained in some mutants.

1.6. Selection of Antibody with Improved Affinity from Libraries

After maturation of the affinity of the constructed libraries for c-Met, the nucleotide sequence of scFv from each clone was analyzed. The nucleotide sequences thus obtained are summarized in Table 3 and were converted into IgG forms. Four antibodies which were respectively produced from clones L3-1, L3-2, L3-3, and L3-5 were used in the subsequent experiments.

TABLE 3 Library Clone constructed CDR Sequence H11-4 CDR-H1 PEYYMS (SEQ ID NO: 22) YC151 CDR-H1 PDYYMS (SEQ ID NO: 23) YC193 CDR-H1 SDYYMS (SEQ ID NO: 24) YC244 CDR-H2 RNNANGNT (SEQ ID NO: 25) YC321 CDR-H2 RNKVNGYT (SEQ ID NO: 26) YC354 CDR-H3 DNWLSY (SEQ ID NO: 27) YC374 CDR-H3 DNWLTY (SEQ ID NO: 28) L1-1 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 29) L1-3 CDR-L1 KSSRSLLSSGNHKNYLA (SEQ ID NO: 30) L1-4 CDR-L1 KSSKSLLASGNQNNYLA (SEQ ID NO: 31) L1-12 CDR-L1 KSSRSLLASGNQNNYLA (SEQ ID NO: 32) L1-22 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 33) L2-9 CDR-L2 WASKRVS (SEQ ID NO: 34) L2-12 CDR-L2 WGSTRVS (SEQ ID NO: 35) L2-16 CDR-L2 WGSTRVP (SEQ ID NO: 36) L3-1 CDR-L3 QQSYSRPYT (SEQ ID NO: 13) L3-2 CDR-L3 GQSYSRPLT (SEQ ID NO: 14) L3-3 CDR-L3 AQSYSHPFS (SEQ ID NO: 15) L3-5 CDR-L3 QQSYSRPFT (SEQ ID NO: 16) L3-32 CDR-L3 QQSYSKPFT (SEQ ID NO: 37)

1.7. Conversion of Selected Antibodies into IgG

Respective polynucleotides encoding heavy chains of the four selected antibodies were designed to have the structure of “EcoRI-signal sequence-VH-NheI-CH-XhoI” (SEQ ID NO: 38). The heavy chains of huAbF46 antibodies were used as they were because their amino acids were not changed during affinity maturation. In the case of the hinge region, however, the U6-HC7 hinge (SEQ ID NO: 57) was employed instead of the hinge of human IgG1. Genes were also designed to have the structure of “EcoRI-signal sequence-VL-BsiWI-CL-XhoI” for the light chain. Polypeptides encoding light chain variable regions of the four antibodies which were selected after the affinity maturation were synthesized in Bioneer. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and DNA fragments having the light chain nucleotide sequences (DNA fragment including L3-1-derived CDR-L3: SEQ ID NO: 58, DNA fragment including L3-2-derived CDR-L3: SEQ ID NO: 59, DNA fragment including L3-3-derived CDR-L3: SEQ ID NO: 60, and DNA fragment including L3-5-derived CDR-L3: SEQ ID NO: 61) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing affinity-matured antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invtrogen) (A), and in another 15 ml tube, 100 ul (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify four affinity-matured antibodies (hereinafter referred to as “huAbF46-H4-A1 (L3-1 origin), huAbF46-H4-A2 (L3-2 origin), huAbF46-H4-A3 (L3-3 origin), and huAbF46-H4-A5 (L3-5 origin),” respectively).

1.8. Construction of Constant Region- and/or Hinge Region-Substituted huAbF46-H4-A1

Among the four antibodies selected in Reference Example 1.7, huAbF46-H4-A1 was found to be the highest in affinity for c-Met and the lowest in Akt phosphorylation and c-Met degradation degree. In the antibody, the hinge region, or the constant region and the hinge region, were substituted.

The antibody huAbF46-H4-A1 (U6-HC7) was composed of a heavy chain including the heavy chain variable region of huAbF46-H4-A1, U6-HC7 hinge, and the constant region of human IgG1 constant region, and a light chain including the light chain variable region of huAbF46-H4-A1 and human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 hinge) was composed of a heavy chain including a heavy chain variable region, a human IgG2 hinge region, and a human IgG1 constant region, and a light chain including the light chain variable region of huAbF46-H4-A1 and a human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 Fc) was composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG2 constant region, and a light chain including the light variable region of huAbF46-H4-A1 and a human kappa constant region. The histidine residue at position 36 on the human kappa constant region of the light chain was changed to tyrosine in all of the three antibodies to increase antibody production.

For use in constructing the three antibodies, a polynucleotide (SEQ ID NO: 63) encoding a polypeptide (SEQ ID NO: 62) composed of the heavy chain variable region of huAbF46-H4-A1, a U6-HC7 hinge region, and a human IgG1 constant region, a polynucleotide (SEQ ID NO: 65) encoding a polypeptide (SEQ ID NO: 64) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG1 region, a polynucleotide (SEQ ID NO: 67) encoding a polypeptide (SEQ ID NO: 66) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 region, and a human IgG2 constant region, and a polynucleotide (SEQ ID NO: 69) encoding a polypeptide (SEQ ID NO: 68) composed of the light chain variable region of huAbF46-H4-A1, with a tyrosine residue instead of histidine at position 36, and a human kappa constant region were synthesized in Bioneer. Then, the DNA fragments having heavy chain nucleotide sequences were inserted into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) while DNA fragments having light chain nucleotide sequences were inserted into a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01) so as to construct vectors for expressing the antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invtrogen) (A), and in another 15 ml tube, 100 ul (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to finally purify three antibodies (huAbF46-H4-A1 (U6-HC7), huAbF46-H4-A1 (IgG2 hinge), and huAbF46-H4-A1 (IgG2 Fc)). Among the three antibodies, huAbF46-H4-A1 (IgG2 Fc) was selected for the following examples, and name as L3-1Y-IgG2.

Reference Example 2: Preparation of a c-Met/EGFR Dual Inhibitor

2.1. Preparation of Anti-EGFR scFv

An anti-EGFR scFv binding to EGFR was prepared by inserting a peptide linker of (GGGGS)₃ (SEQ ID NO: 132) between a heavy chain variable region of SEQ ID NO: 115 and a light chain variable region of SEQ ID NO: 116. In particular, the DNA sequence encoding a (GGGGS)₃ linker peptide was added to the DNA sequence (SEQ ID NO: 119) encoding the heavy chain variable region (SEQ ID NO: 115) and the DNA sequence (SEQ ID NO: 120) encoding the light chain variable region (SEQ ID NO: 116) of a humanized anti-EGFR antibody using automatic gene synthesis (Bioneer Inc.) to synthesize a DNA fragment encoding a scFv of the anti-EGFR antibody. An anti-EGFR scFv prepared from the synthesized DNA fragment was named “anti-EGFR antibody E-2”.

The amino acid sequences of the heavy chain variable region and the light chain variable region of the prepared anti-EGFR scFv, and coding nucleotide sequences thereof are summarized in Table 4, as follows (wherein the sequences marked in bold type indicate CDRs, i.e., CDR-H1, CDR-H2, and CDR-H3, or CDR-L1, CDR-L2, and CDR-L3, in sequence):

TABLE 4 Heavy chain Light chain variable region variable region of anti-EGFR of anti-EGFR antibody E-2 antibody E-2 Amino EVQLLESGGGLVQPGG QSVLTQPPSASGTPGQ acid SLRLSCAASGFTFSNY RVTISCTGSSSNIGNN sequence DMSWVRQAPGKGLEWV DVSWYQQLPGTAPKLL SGISHSSGSKYYADSV IYDDNKRPSGVPDRFS KGRFTISRDNSKNTLY GSKSGTSASLAISGLR LQMNSLRAEDTAVYYC SEDEADYYCGSWDASL AKDATPRPLKPFDYWG NAYVFGGGTKLTVLG QGTLVTVSS (SEQ ID NO: 116) (SEQ ID NO: 115) Coding GAGGTGCAGCTGTTGG CAGTCTGTGCTGACTC nucleotide AGTCTGGGGGAGGCTT AGCCACCCTCAGCGTC sequence GGTACAGCCTGGGGGG TGGGACCCCCGGGCAG TCCCTGAGACTCTCCT AGGGTCACCATCTCTT GTGCAGCCTCTGGATT GTACTGGCTCTTCATC CACCTTTAGCAATTAT TAATATTGGCAATAAT GATATGAGCTGGGTCC GATGTCTCCTGGTACC GCCAGGCTCCAGGGAA AGCAGCTCCCAGGAAC GGGGCTGGAGTGGGTC GGCCCCCAAACTCCTC TCAGGGATCTCTCATA ATCTATGATGATAATA GTAGTGGTAGTAAATA AGCGGCCAAGCGGGGT TTACGCTGATTCTGTA CCCTGACCGATTCTCT AAAGGTCGGTTCACCA GGCTCCAAATCTGGCA TCTCCAGAGACAATTC CCTCAGCCTCCCTGGC CAAGAACACGCTGTAT CATCAGTGGGCTCCGG CTGCAAATGAACAGCC TCCGAGGATGAGGCTG TGAGAGCCGAGGACAC ATTATTACTGTGGTTC GGCCGTGTATTACTGT TTGGGATGCTAGCCTG GCGAAAGATGCTACTC AATGCTTATGTCTTCG CGCGTCCGCTGAAGCC GCGGAGGCACCAAGCT TTTCGACTACTGGGGC GACGGTCCTAGGC CAGGGTACACTGGTCA (SEQ ID NO: 120) CCGTGAGCTCA (SEQ ID NO: 119)

A modified anti-EGFR scFv (heavy chain variable region: SEQ ID NO: 117 and light chain variable region: SEQ ID NO: 118) was prepared as described above, with the exception that the amino acid, G, at 44^(th) position of the heavy chain variable region (SEQ ID NO: 115) was substituted with C, and the amino acid, G, at 100^(th) position of the light chain variable region (SEQ ID NO: 116) was substituted with C. The amino acid location within the antibody complies with kabat numbering system. Such modifications (substitutions) can increase the stability of the anti-EGFR scFv.

<SEQ ID NO: 117: heavy chain variable region of modified anti-EGFR antibody E-2>

EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKCLEWVSGISH SSGSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDATPRPLKPFDY WGQGTLVTVSS

(wherein the sequences marked in bold type indicate CDRs, i.e., CDR-H1, CDR-H2, and CDR-H3, in sequence)

<SEQ ID NO: 118: light chain variable region of modified anti-EGFR antibody E-2>

QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNDVSWYQQLPGTAPKLLIYDDNK RPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDASLNAYVFGCGTKLTVLG

(wherein the sequences marked in bold type indicate CDRs, i.e., CDR-L1, CDR-L2, and CDR-L3, in sequence)

The obtained modified anti-EGFR scFv (including SEQ ID NO: 117 and SEQ ID NO: 118) was used to manufacture the following bispecific antibodies.

2.2. Preparation of a c-Met/EGFR Dual Inhibitor (Anti-c-Met/Anti-EGFR Bispecific Antibody)

The modified anti-EGFR scFv (including SEQ ID NO: 117 and SEQ ID NO: 118) prepared in the above Reference Example 2.1 was fused at the c-terminus of the Fc portion of the anti-c-Met antibody L3-1Y-IgG2 prepared in the above reference example 1. The fusion procedures are as follows.

A DNA segment having a base sequence (SEQ ID NO: 66) corresponding to the heavy chain of the anti-c-Met antibody L3-1Y-IgG2 prepared in above reference example 1 was inserted into a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01) which is included in OptiCHO™ Antibody Express Kit (Cat no. 12762-019) by Invitrogen Inc., and a DNA segment having a base sequence (SEQ ID NO: 68) corresponding to the light chain of the anti-c-Met antibody L3-1Y-IgG2 was inserted into a pOptiVEC™-TOPO TA Cloning Kit. Thereafter, the anti-EGFR scFv coding DNA prepared in Example 1 was fused at the c-terminus of the Fc portion of L3-1Y-IgG2 inserted into pcDNA™3.3, using the coding DNA sequence of a linker peptide having 10 amino acid lengths consisting of (GGGGS)₂ (SEQ ID NO: 133), to construct vectors for the expression of bispecific antibodies.

The constructed vectors were each amplified using Qiagen Maxiprep kit (Cat no. 12662 and transient expression of the constructed vectors was obtained using the Freestyle™ MAX 293 Expression System (invitrogen). The cell line used was 293 F cells, which were cultured in a suspension culture using FreeStyle™ 293 Expression Medium. One day before the transient expression, the cells were prepared at a concentration of 5×10⁵ cells/ml and after 24 hours, their transient expression started when the number of the cells reached 1×10⁶ cells/ml. Transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen). DNA was prepared in a 15-ml tube in a ratio of heavy chain DNA:light chain DNA=3:2 and mixed with 2 ml of OptiPro™ SFM (invtrogen) (A), and 100 μl of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed in another 15-ml tube (B), and after (A) and (B) were mixed and incubated for 15 min., the mixture solution was then slowly mixed into the cells which were prepared one day before. After the transfection was complete, the cells were cultured in a 37° C., 80% humidity, 8% CO₂, 130 rpm incubator for 5 days.

The cultured cells were centrifuged to obtain 100 ml of supernatants, which were then purified using AKTA Prime (GE healthcare). The culture was flowed at a flow rate of 5 ml/min. onto the AKTA Prime installed with Protein A column (GE healthcare, 17-0405-03) to perform elution using an IgG elution buffer (Thermo Scientific, 21004). The buffer was replaced by a PBS buffer to finally obtain purified a c-Met/EGFR dual inhibitor (an anti-c-Met/anti-EGFR bispecific antibody).

The prepared anti-c-Met/anti-EGFR bispecific antibody in which the modified anti-EGFR scFv is fused at the c-terminal of L3-1Y-IgG2 was named ME22S.

Example 1: Measurement of the Level of IGF-1R Protein in Lung Cancer Cells and Gastric Cancer Cells

In the lung cancer cell lines H820 (ATCC, HTB-181) and HCC827 (ATCC, CRL-2868), and gastric cancer cell lines SNU5 (ATCC, CRL-5973), SNU668 (KCLB, 00668), and MKN45(JCRB 0254), the level of IGF-1R protein was measured.

In particular, each of the cell lines was seeded on a 60 mm plate at the amount of 2×10⁵ cells/ml, and 48 hours after, collected and lysed using lysis buffer (lysis-M, Roche), to extract proteins. Thereafter, the amounts of IGF-1R and GAPDH proteins were measured by western blotting (antibodies used: products of Cell signaling).

The results, shown in the upper part of FIG. 1, indicate that for lung cancer cells, the level of IGF-1R is relatively high in the HCC827 line, whereas it is considerably low in H820 cell line. For gastric cancer cells, IGF-1R is nearly absent in the SNU5 cell line, whereas it is present at a relatively high levels in the SNU668 and MKN45 cell lines.

The five cell lines were co-administered with ME22S prepared in Reference 2 and IGF-1R inhibitor, linsitinib (Selleck Chemical S1091), to measure the cell proliferation inhibition effect and examine the correlation between the effect or treatment and the level of IGF-1R.

For the cell proliferation assays, each of the cell lines were seeded on 96-well plate at the amount of 5×10³ cells/well, and 24 hours later were treated with the ME22S antibody at a concentration of 0 nM, 0.08 nM, 0.4 nM, 2 nM, or 1.0 nM, in presence or absence of linsitinib (10 μM). 72 hours after the treatment with the antibody and/or the inhibitor drug, the number of living cells was counted using a CellTiter Glo assay (Promega, G7573). This assay measures the amount of ATP which reflects metabolism of living cells, thereby counting the number of living cells.

The results, shown in bottom part of FIG. 1, indicate that in cell lines H820 and SNU5 which have low levels of IGF-1R, there is no or only slight effect of co-administration of ME22S and linsitinib, whereas in cell lines HCC827, SNU668, and MKN45 which have high levels of IGF-1R, there is considerable effect of the co-administration. These results indicate that when IGF-1R is present at a high level, a considerable synergistic effect can be achieved by co-administration of a c-Met/EGFR dual inhibitor and an IGF-1R inhibitor.

Example 2: Examination of Effect of Co-Administration of ME22S and an IGF-1R Inhibitor in Gastric Cancer Cells

As identified from Example 1, IGF-1R is not found in gastric cell line SNU5 and the level of EGFR in gastric cell line SNU668 is higher than that of the gastric cell lines. In these two cell lines, the cell proliferation inhibition effect of co-administration of ME22S and an IGF-1R inhibitor linsitinib was examined.

In particular, SNU5 or SNU668 cells were seeded on 96-well plates at the amount of 5000 cells/well (medium used: RPMI (GIBCO); culture conditions: 5% CO₂ and 37° C.), and 24 hours after, treated with ME22S and linsitinib. 72 hours after the co-treatment, the cell number was measured by CellTiter Glo assay (Promega, G7573). Linsitinib was treated at a fixed concentration of 10 μM, and ME22S was treated at the 1/5 diluted concentration starting from 60 nM (see FIG. 2). For comparison, cells were treated with the same concentrations of ME22S in the absence of linsitinib.

The results, shown in FIG. 2, indicate that there was no synergistic effect by co-administration of ME22S and linsitinib in cell line SNU5, on which ME22S has a cell proliferation inhibition effect even when it is administered alone; whereas in cell line SNU668 on which ME22S does not exhibit a cell proliferation inhibition effect when it is administered alone, a synergistic anticancer effect can be achieved by co-administration of ME22S and linsitinib.

Example 3: Examination of Effect of Co-Administration of ME22S and an IGF-1R Inhibitor in Lung Cancer Cells

In order to examine a synergistic effect of co-administration of ME22S and an IGF-1R inhibitor in a lung cancer cell line on which ME22S does not exhibit its effect (e.g., cell proliferation inhibition effect) when it is administered alone, the HCC827 cell line was used.

HCC827 cell line were seeded at 5000 cells/well on a 96-well plate (medium: RPMI (GIBCO); culture condition: 5% CO₂ and 37° C.), and 24 hours after, treated with ME22S and linsitinib together. 72 hours after the co-treatment, the number of living cells was counted by CellTiter Glo assay (Promega, G7573). Linsitinib was treated at a fixed concentration of 10 μM, and ME22S was treated at the 1/5 diluted concentration starting from 150 nM (see FIG. 3A). For comparison, the same experiment was performed except cells were treated with ME22S alone at the 1/5 diluted concentration starting from 150 nM or linsitinib alone at the concentration of 10 μM.

The results from these experiments are shown in FIGS. 3A and 3B. FIG. 3A shows degrees of cancer cell proliferation depending on the concentrations of ME22S. FIG. 3B shows degrees of cancer cell proliferation at the concentrations of 30 nM of ME22S and 10 μM of linsitinib. As shown in FIGS. 3A and 3B, in HCC827 cells, a cancer cell proliferation inhibition effect is observed when ME22S and linsitinib are administered together, whereas it is not observed when ME22S or linsitinib is administered alone.

Example 4: Examination II of Effect of Co-Administration of ME22S and an IGF-1R Inhibitor in Gastric Cancer Cells

In order to examine a synergistic effect of co-administration of ME22S and an IGF-1R inhibitor in a gastric cancer cell line on which ME22S exhibits its effect (cell proliferation inhibition effect) even when it is administered alone, the MKN45 cell line was employed.

MKN45 cell line were seeded at 5000 cells/well on a 96-well plate (medium: RPMI (GIBCO); culture condition: 5% CO₂ and 37° C.), and 24 hours after, treated with ME22S and linsitinib together. 72 hours after the co-treatment, the number of living cells was counted by CellTiter Glo assay (Promega, G7573). Linsitinib was treated at a fixed concentration of 10 μM, and ME22S was treated at the 1/5 diluted concentration starting from relatively low concentration of 10 nM (see FIG. 4A) since ME22S alone can inhibit proliferation of the MKN45 cell line. For comparison, the same experiment was performed except the cells were treated with ME22S alone at the 1/5 diluted concentration starting from 10 nM or linsitinib alone at the concentration of 10 μM.

The results from these experiments are shown in FIGS. 4A and 4B. FIG. 4A shows degrees of cancer cell proliferation depending on the concentration of ME22S. FIG. 4B shows degrees of cancer cell proliferation at the concentrations of 0.4 nM of ME22S and 10 μM of linsitinib. As shown in FIGS. 4A and 4B, in MKN45 cells, increased cancer cell proliferation inhibition is observed when ME22S and linsitinib are administered together, compared to when ME22S or linsitinib is administered alone. In addition, the increased cancer cell proliferation inhibition can be achieved at the relatively low concentration (such as about 0.4 nM) of ME22S when ME22S is administered together with linsitinib, indicating that the dose of each drug (i.e., c-Met/EGFR dual inhibitor ME22S or IGF-1R inhibitor linsitinib) can be reduced by co-administration thereof.

Example 5: Examination of Effect of Co-Administration of ME22S and an IGF-1R Inhibitor in Anti-c-Met Antibody Resistant Gastric Cancer Cells

Anti-c-Met antibody resistance acquired gastric cancer cells, obtained by long term-administering anti-c-Met antibody L3-1Y/IgG2 prepared in Reference Example 1 to MKN45 cells (JCRB 0254) on which anti-c-Met antibody L3-1Y/IgG2 exhibits its effect (e.g., cancer cell proliferation inhibition effect) when it is administered alone, were used for the following experiment.

To obtain anti-c-Met antibody resistant MKN45 gastric cancer cells, MKN45 gastric cancer cells were administered with L3-1Y/IgG2 for at least 3 months by increasing the administration concentration. The amount of L3-1Y/IgG2 administered was increased from 1 μg/ml to 10 μg/ml, until resistance to L3-1Y/IgG2 is induced. The obtained L3-1Y/IgG2 resistant clones were named as MKN45re#1 and MKN45re#24, respectively.

It was confirmed that the obtained anti-c-Met antibody (L3-1Y/IgG2) resistant MKN45 gastric cancer cell line is also resistant to ME22S.

Each of the L3-1Y/IgG2 resistant MKN45re#1 and MKN45re#24 cell lines were seeded on a 96-well plate at the amount of 5000 cells/well (medium: RPMI (GIBCO); culture condition: 5% CO₂ and 37° C.), and 24 hours after, treated with ME22S and linsitinib together. 72 hours after the co-treatment, the number of living cells was counted by CellTiter Glo assay (Promega, G7573). Linsitinib was treated at a fixed concentration of 10 μM (MKN45re#1) or 2.5 μM (MKN45re#24), and ME22S was treated at the 1/5 diluted concentration starting from 10 nM (see FIG. 5). For comparison, the same experiment was preformed except that cells were treated with ME22S alone at the 1/5 diluted concentration starting from 10 nM.

The results from this experiment are shown in FIG. 5. As shown in FIG. 5, in the L3-1Y/IgG2 resistance acquired MKN45 cell line, the administration of ME22S alone has no cancer cell proliferation inhibition effect (instead, leads to increase in cancer cell proliferation); whereas the combined administration of ME22S and linsitinib causes considerable cancer cell proliferation inhibition. These results indicate that the anticancer effect can be restored by combined administration of ME22S and linsitinib, even on a cell line on which ME22S alone loses its anticancer effect.

Example 6: Examination of Effect of Co-Administration of ME22S and an IGF-1R Inhibitor in Anti-c-Met Antibody Resistant Gastric Cancer Cells

Changes in the activities (e.g., phosphorylation) of downstream proteins upon co-administration of ME22S and linsitinib to the L3-1Y/IgG2 resistant cell line MKN45re#1 prepared in Example 5 were measured. In particular, the cell line was seeded on 60 mm plate at the amount of 2×10⁵ cells/ml, and 24 hours after, treated with antibody ME22S (10 nM) provided in serum-free condition and/or linsitinib (10 μM) for 30 minutes. Thereafter, the phosphorylation degrees of c-Met and AKT were measured by western blotting.

The results are shown in FIG. 6. As shown in FIG. 6, when the L3-1Y/IgG2 resistant cell line MKN45re#1 is treated with ME22S and linsitinib, the activity of p-AKT which is a downstream protein related to tumorigenesis is inhibited.

Example 7: Measurement of Degree of IGF-1R Activation in a Lung Cancer Cell Line Co-Treated with ME22S and Linsitinib

It is possible to detect an activated site of IGF-1R using an antibody for immunoblotting of p-IGF-1R beta.

Lung cancer cell line HCC827 (ATCC, CRL-2868) was seeded on 60 mm plate at the amount of 2×10⁵ cells/ml, and 24 hours after, treated with antibody ME22S (10 nM) provided in serum-free condition and/or linsitinib (10 μM) for 30 minutes. Thereafter, the phosphorylation degree of IGF-1R was measured by western blotting using an anti-p-IGF1R beta antibody (Cell signaling).

The results are shown in FIG. 7. As shown in FIG. 7, the co-treatment of ME22S and linsitinib leads to a decrease in the level of p-IGF-1R, indicating that the simultaneous inhibition of all of c-Met, EGFR and IGF-1R is more effective compared to the inhibition of one or two of c-Met, EGFR and IGF-1R.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. 

What is claimed is:
 1. A method of treating a cancer in a subject, comprising co-administering a c-Met/EGFR dual inhibitor and an insulin-like growth factor 1 receptor (IGF-1R) inhibitor to the subject, wherein the c-Met/EGFR dual inhibitor comprises: 1) an anti-c-Met antibody or an antigen-binding fragment thereof, and 2) an anti-EGFR antibody, an antigen-binding fragment thereof, or an anti-EGFR DARPin, wherein the anti-c-Met antibody or an antigen-binding fragment thereof recognizes or binds to a polypeptide comprising 5 to 19 contiguous amino acid residues within SEQ ID NO: 71, wherein the polypeptide comprises at least SEQ ID NO: 73, and the anti-EGFR antibody, an antigen-binding fragment thereof, or an anti-EGFR DARPin comprises: a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 109, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 110, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 111, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 112, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 113, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 114, b) at least one selected from the group consisting of cetuximab, panitumumab, an anti-EGFR antibody or an antigen-binding fragment thereof comprising a heavy chain variable region of SEQ ID NO: 121 and a light chain variable region of SEQ ID NO: 123, and an anti-EGFR antibody or an antigen-binding fragment thereof comprising a heavy chain variable region of SEQ ID NO: 125 and a light chain variable region of SEQ ID NO: 126, or c) 1 to 10 anti-EGFR DARPin units, each of which is independently selected from the group consisting of SEQ ID NOs: 127 to 130, and wherein the cancer is gastric cancer or lung cancer that is resistant to treatment with an anti-c-Met antibody or a c-Met/EGFR dual inhibitor in the absence of an IGF-1R inhibitor.
 2. The method of claim 1, wherein the c-Met/EGFR dual inhibitor and an IGF-1R inhibitor are in a single or mixed formulation, or each of the c-Met/EGFR dual inhibitor and an IGF-1R inhibitor are administered in separate compositions, simultaneously or sequentially in any order.
 3. The method of claim 1, wherein the anti-c-Met antibody or an antigen-binding fragment thereof comprises: a CDR-H1 comprising SEQ ID NO: 4; a CDR-H2 comprising SEQ ID NO: 2, SEQ ID NO: 5, or an amino acid sequence comprising 8-19 consecutive amino acids within SEQ ID NO: 2 comprising the 3^(rd) to 10^(th) positions of SEQ ID NO: 2; a CDR-H3 comprising SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 85, or an amino acid sequence comprising 6-13 consecutive amino acids within SEQ ID NO: 85 comprising the 1^(st) to 6^(th) positions of the amino acid sequence of SEQ ID NO: 85, a CDR-L1 comprising SEQ ID NO: 7, a CDR-L2 comprising SEQ ID NO: 8, and a CDR-L3 comprising SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 86, or 9-17 consecutive amino acids within SEQ ID NO: 89 comprising the 1^(st) to 9^(th) positions of SEQ ID NO:
 89. 4. The method of claim 3, wherein the anti-c-Met antibody or an antigen-binding fragment thereof comprises: a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 1, 22, 23, or 24, a CDR-H2 comprising SEQ ID NO: 2, 25, or 26, and a CDR-H3 comprising SEQ ID NO: 3, 27, 28, or 85; a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 10, 29, 30, 31, 32, 33, or 106, a CDR-L2 comprising SEQ ID NO: 11, 34, 35, or 36, and a CDR-L3 comprising SEQ ID NO: 12, 13, 14, 15, 16, 37, 86, or 89; or a combination thereof.
 5. The method of claim 3, wherein the anti-c-Met antibody or an antigen-binding fragment thereof comprises: a heavy chain variable region comprising SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94; a light chain variable region comprising SEQ ID NO: 131, 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99, or 107; or a combination thereof.
 6. The method of claim 3, wherein the anti-c-Met antibody comprises: a heavy chain comprising SEQ ID NO: 62, an amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62, SEQ ID NO: 64, an amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64, SEQ ID NO: 66, or an amino acid sequence from the 18th to 460^(th) positions of SEQ ID NO: 66; and a light chain comprising SEQ ID NO: 68, an amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68, SEQ ID NO: 70, an amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70, or SEQ ID NO:
 108. 7. The method of claim 1, wherein the anti-EGFR antibody or an antigen-binding fragment thereof comprises: a heavy chain variable region comprising SEQ ID NO: 115, 117, 121, or 125; a light chain variable region comprising SEQ ID NO: 116, 118, 123, or 126; or a combination thereof.
 8. The method of claim 1, wherein the c-Met/EGFR dual inhibitor comprises: 1) an anti-c-Met antibody and 2) an antigen-binding fragment of an anti-EGFR antibody or an anti-EGFR DARPin comprising 1 to 10 anti-EGFR DARPin units, each of which is independently selected from the group consisting of SEQ ID NOs: 127 to 130, wherein the antigen-binding fragment of an anti-EGFR antibody or the anti-EGFR DARPin is linked to the C-terminus of the anti-c-Met antibody.
 9. The method of claim 1, wherein the IGF-1R inhibitor comprises at least one selected from the group consisting of an antibody, an aptamer, siRNA, shRNA, microRNA, a small molecule IGF-1R inhibitor, and a pharmaceutically acceptable salt thereof.
 10. The method of claim 1, wherein the IGF-1R inhibitor is at least one selected from the group consisting of linsitinib, NVP-AEW541, GSK1904529A, NVP-ADW742, BMS-536924, figitumumab, cixutumumab, dalotuzumab, R1507, XL-228, INSM-18, BMS-754807, AG-1024, GSK1838705A, PQ 401, and pharmaceutically acceptable salts thereof.
 11. The method of claim 1, wherein the cancer is characterized by overexpression of c-Met. 